Single-chain multivalent binding proteins with effector function

ABSTRACT

Multivalent binding peptides, including bi-specific binding peptides, having immunoglobulin effector function are provided, along with encoding nucleic acids, vectors and host cells as well as methods for making such peptides and methods for using such peptides to treat or prevent a variety of diseases, disorders or conditions, as well as to ameliorate at least one symptom associated with such a disease, disorder or condition.

FIELD OF THE INVENTION

The invention relates generally to the field of multivalent bindingmolecules and therapeutic applications thereof.

The sequence listing is being submitted as a text file and as a PDF filein compliance with applicable requirements for electronic filing. Thesequence listing was created on Jun. 12, 2007. The sequence listing isincorporated herein by reference in its entirety.

BACKGROUND

In a healthy mammal, the immune system protects the body from damagefrom foreign substances and pathogens. In some instances though, theimmune system goes awry, producing traumatic insult and/or disease. Forexample, B-cells can produce antibodies that recognize self-proteinsrather than foreign proteins, leading to the production of theautoantibodies characteristic of autoimmune diseases such as lupuserythematosus, rheumatoid arthritis, and the like. In other instances,the typically beneficial effect of the immune system in combatingforeign materials is counterproductive, such as following organtransplantation. The power of the mammalian immune system, and inparticular the human immune system, has been recognized and efforts havebeen made to control the system to avoid or ameliorate the deleteriousconsequences to health that result either from normal functioning of theimmune system in an abnormal environment (e.g., organ transplantation)or from abnormal functioning of the immune system in an otherwiseapparently normal environment (e.g., autoimmune disease progression).Additionally, efforts have been made to exploit the immune system toprovide a number of target-specific diagnostic and therapeuticmethodologies, relying on the capacity of antibodies to specificallyrecognize and bind antigenic targets with specificity.

One way in which the immune system protects the body is by production ofspecialized cells called B lymphocytes or B-cells. B-cells produceantibodies that bind to, and in some cases mediate destruction of, aforeign substance or pathogen. In some instances though, the humanimmune system, and specifically the B lymphocytes of the human immunesystem, go awry and disease results. There are numerous cancers thatinvolve uncontrolled proliferation of B-cells. There are also numerousautoimmune diseases that involve B-cell production of antibodies that,instead of binding to foreign substances and pathogens, bind to parts ofthe body. In addition, there are numerous autoimmune and inflammatorydiseases that involve B-cells in their pathology, for example, throughinappropriate B-cell antigen presentation to T-cells or through otherpathways involving B-cells. For example, autoimmune-prone mice deficientin B-cells do not develop autoimmune kidney disease, vasculitis orautoantibodies. (Shlomchik et al., J Exp. Med. 1994, 180:1295-306).Interestingly, these same autoimmune-prone mice which possess B-cellsbut are deficient in immunoglobulin production, do develop autoimmunediseases when induced experimentally (Chan et al., J Exp. Med. 1999,189:1639-48), indicating that B-cells play an integral role indevelopment of autoimmune disease.

B-cells can be identified by molecules on their cell surface. CD20 wasthe first human B-cell lineage-specific surface molecule identified by amonoclonal antibody. It is a non-glycosylated, hydrophobic 35 kDa B-celltransmembrane phosphoprotein that has both its amino and carboxy endssituated inside the cell. Einfeld et al., EMBO J. 1988, 7:711-17. CD20is expressed by all normal mature B-cells, but is not expressed byprecursor B-cells or plasma cells. Natural ligands for CD20 have notbeen identified, and the function of CD20 in B-cell biology is stillincompletely understood.

Another B-cell lineage-specific cell surface molecule is CD37. CD37 is aheavily glycosylated 40-52 kDa protein that belongs to the tetraspanintransmembrane family of cell surface antigens. It traverses the cellmembrane four times forming two extracellular loops and exposing itsamino and carboxy ends to the cytoplasm. CD37 is highly expressed onnormal antibody-producing (sIg+)B-cells, but is not expressed onpre-B-cells or plasma cells. The expression of CD37 on resting andactivated T cells, monocytes and granulocytes is low and there is nodetectable CD37 expression on NK cells, platelets or erythrocytes. See,Belov et al., Cancer Res., 61(11):4483-4489 (2001); Schwartz-Albiez etal., J. Immunol., 140(3): 905-914 (1988); and Link et al., J. Immunol.,137(9): 3013-3018 (1988). Besides normal B-cells, almost allmalignancies of B-cell origin are positive for CD37 expression,including CLL, NHL, and hairy cell leukemia (Moore, et al. 1987; Mersonand Brochier 1988; Faure, et al. 1990). CD37 participates in regulationof B-cell function, since mice lacking CD37 were found to have lowlevels of serum IgG1 and to be impaired in their humoral response toviral antigens and model antigens. It appears to act as a nonclassicalcostimulatory molecule or by directly influencing antigen presentationvia complex formation with MHC class II molecules. See Knobeloch et al.,Mol. Cell. Biol., 20(15):5363-5369 (2000).

Research and drug development has occurred based on the concept thatB-cell lineage-specific cell surface molecules such as CD37 and CD20 canthemselves be targets for antibodies that would bind to, and mediatedestruction of, cancerous and autoimmune disease-causing B-cells thathave CD37 and CD20 on their surfaces. Termed “immunotherapy,” antibodiesmade (or based on antibodies made) in a non-human animal that bind toCD37 or CD20 were given to a patient to deplete cancerous or autoimmunedisease-causing B-cells.

Monoclonal antibody technology and genetic engineering methods havefacilitated development of immunoglobulin molecules for diagnosis andtreatment of human diseases. The domain structure of immunoglobulins isamenable to engineering, in that the antigen binding domains and thedomains conferring effector functions may be exchanged betweenimmunoglobulin classes and subclasses. Immunoglobulin structure andfunction are reviewed, for example, in Harlow et al., Eds., Antibodies:A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, ColdSpring Harbor (1988). An extensive introduction as well as detailedinformation about all aspects of recombinant antibody technology can befound in the textbook “Recombinant Antibodies” (John Wiley & Sons, NY,1999). A comprehensive collection of detailed antibody engineering labProtocols can be found in R. Kontermann and S. Dübel (eds.), “TheAntibody Engineering Lab Manual” (Springer Verlag, Heidelberg/New York,2000).

An immunoglobulin molecule (abbreviated Ig), is a multimeric protein,typically composed of two identical light chain polypeptides and twoidentical heavy chain polypeptides (H₂L₂) that are joined into amacromolecular complex by interchain disulfide bonds, i.e., covalentbonds between the sulfhydryl groups of neighboring cysteine residues.Five human immunoglobulin classes are defined on the basis of theirheavy chain composition, and are named IgG, IgM, IgA, IgE, and IgD. TheIgG-class and IgA-class antibodies are further divided into subclasses,namely, IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2, respectively.Intrachain disulfide bonds join different areas of the same polypeptidechain, which results in the formation of loops that, along with adjacentamino acids, constitute the immunoglobulin domains. At theamino-terminal portion, each light chain and each heavy chain has asingle variable region that shows considerable variation in amino acidcomposition from one antibody to another. The light chain variableregion, V_(L), has a single antigen-binding domain and associates withthe variable region of a heavy chain, V_(H) (also containing a singleantigen-binding domain), to form the antigen binding site of theimmunoglobulin, the Fv.

In addition to variable regions, each of the full-length antibody chainshas a constant region containing one or more domains. Light chains havea constant region containing a single domain. Thus, light chains haveone variable domain and one constant domain. Heavy chains have aconstant region containing several domains. The heavy chains in IgG,IgA, and IgD antibodies have three domains, which are designated C_(H1),C_(H2), and C_(H3); the heavy chains in IgM and IgE antibodies have fourdomains, C_(H1), C_(H2), C_(H3) and C_(H4). Thus, heavy chains have onevariable domain and three or four constant domains. Noteworthy is theinvariant organization of these domains in all known species, with theconstant regions, containing one or more domains, being located at ornear the C-terminus of both the light and heavy chains of immunoglobulinmolecules, with the variable domains located towards the N-termini ofthe light and heavy chains. Immunoglobulin structure and function arereviewed, for example, in Harlow et al., Eds., Antibodies: A LaboratoryManual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor(1988).

The heavy chains of immunoglobulins can also be divided into threefunctional regions: the Fd region (a fragment comprising V_(H) andC_(H1), i.e., the two N-terminal domains of the heavy chain), the hingeregion, and the Fc region (the “fragment crystallizable” region). The Fcregion contains the domains that interact with immunoglobulin receptorson cells and with the initial elements of the complement cascade. Thus,the Fc region or fragment is generally considered responsible for theeffector functions of an immunoglobulin, such as ADCC(antibody-dependent cell-mediated cytotoxicity), CDC(complement-dependent cytotoxicity) and complement fixation, binding toFc receptors, greater half-life in vivo relative to a polypeptidelacking an F_(C) region, protein A binding, and perhaps even placentaltransfer. Capon et al., Nature, 337: 525-531, (1989). Further, apolypeptide containing an Fc region allows fordimerization/multimerization of the polypeptide. These terms are alsoused for analogous regions of the other immunoglobulins.

Although all of the human immunoglobulin isotypes contain a recognizablestructure in common, each isotype exhibits a distinct pattern ofeffector function. IgG, by way of nonexhaustive example, neutralizestoxins and viruses, opsonizes, fixes complement (CDC) and participatesin ADCC. IgM, in contrast, neutralizes blood-borne pathogens andparticipates in opsonization. IgA, when associated with its secretorypiece, is secreted and provides a primary defense to microbial infectionvia the mucosa; it also neutralizes toxins and supports opsonization.IgE mediates inflammatory responses, being centrally involved in therecruitment of other cells needed to mount a full response. IgD is knownto provide an immunoregulatory function, controlling the activation of Bcells. These characterizations of isotype effector functions provide anon-comprehensive illustration of the differences that can be foundamong human isotypes.

The hinge region, found in IgG, IgA, IgD, and IgE class antibodies, actsas a flexible spacer, allowing the Fab portion to move freely in space.In contrast to the constant regions, the hinge domains are structurallydiverse, varying in both sequence and length among immunoglobulinclasses and subclasses. For example, the length and flexibility of thehinge region varies among the IgG subclasses. The hinge region of IgG1encompasses amino acids 216-231 and, because it is freely flexible, theFab fragments can rotate about their axes of symmetry and move within asphere centered at the first of two inter-heavy chain disulfide bridges.IgG2 has a shorter hinge than IgG1, with 12 amino acid residues and fourdisulfide bridges. The hinge region of IgG2 lacks a glycine residue, isrelatively short, and contains a rigid poly-proline double helix,stabilized by extra inter-heavy chain disulfide bridges. Theseproperties restrict the flexibility of the IgG2 molecule. IgG3 differsfrom the other subclasses by its unique extended hinge region (aboutfour times as long as the IgG1 hinge), containing 62 amino acids(including 21 prolines and 11 cysteines), forming an inflexiblepoly-proline double helix. In IgG3, the Fab fragments are relatively faraway from the Fc fragment, giving the molecule a greater flexibility.The elongated hinge in IgG3 is also responsible for its higher molecularweight compared to the other subclasses. The hinge region of IgG4 isshorter than that of IgG1 and its flexibility is intermediate betweenthat of IgG1 and IgG2. The flexibility of the hinge regions reportedlydecreases in the order IgG3>IgG1>IgG4>IgG2. The four IgG subclasses alsodiffer from each other with respect to their effector functions. Thisdifference is related to differences in structure, including differenceswith respect to the interaction between the variable region, Fabfragments, and the constant Fc fragment.

According to crystallographic studies, the immunoglobulin hinge regioncan be further subdivided functionally into three regions: the upperhinge region, the core region, and the lower hinge region. Shin et al.,1992 Immunological Reviews 130:87. The upper hinge region includes aminoacids from the carboxyl end of C_(H1) to the first residue in the hingethat restricts motion, generally the first cysteine residue that formsan interchain disulfide bond between the two heavy chains. The length ofthe upper hinge region correlates with the segmental flexibility of theantibody. The core hinge region contains the inter-heavy chain disulfidebridges, and the lower hinge region joins the amino terminal end of theC_(H2) domain and includes residues in C_(H2). Id. The core hinge regionof human IgG1 contains the sequence Cys-Pro-Pro-Cys which, whendimerized by disulfide bond formation, results in a cyclic octapeptidebelieved to act as a pivot, thus conferring flexibility. The hingeregion may also contain one or more glycosylation sites, which include anumber of structurally distinct types of sites for carbohydrateattachment. For example, IgA1 contains five glycosylation sites within a17-amino-acid segment of the hinge region, conferring resistance of thehinge region polypeptide to intestinal proteases, considered anadvantageous property for a secretory immunoglobulin.

Conformational changes permitted by the structure and flexibility of theimmunoglobulin hinge region polypeptide sequence may also affect theeffector functions of the Fc portion of the antibody. Three generalcategories of effector functions associated with the Fc region include(1) activation of the classical complement cascade, (2) interaction witheffector cells, and (3) compartmentalization of immunoglobulins. Thedifferent human IgG subclasses vary in the relative efficacies withwhich they fix complement, or activate and amplify the steps of thecomplement cascade. See, e.g., Kirschfink, 2001 Immunol. Rev. 180:177;Chakraborti et al., 2000 Cell Signal 12:607; Kohl et al., 1999 Mol.Immunol. 36:893; Marsh et al., 1999 Curr. Opin. Nephrol. Hypertens.8:557; Speth et al., 1999 Wien Klin. Wochenschr. 111:378.

Exceptions to the H₂L₂ structure of conventional antibodies occur insome isotypes of the immunoglobulins found in camelids (camels,dromedaries and llamas; Hamers-Casterman et al., 1993 Nature 363:446;Nguyen et al., 1998 J. Mol. Biol 275:413), nurse sharks (Roux et al.,1998 Proc. Nat. Acad. Sci. USA 95:11804), and in the spotted ratfish(Nguyen, et al., 2002 Immunogenetics 54(1):39-47). These antibodies canapparently form antigen-binding regions using only heavy chain variableregion, i.e., these functional antibodies are homodimers of heavy chainsonly (referred to as “heavy-chain antibodies” or “HCAbs”). Despite theadvantages of antibody technology in disease diagnosis and treatment,there are some disadvantageous aspects of developing whole-antibodytechnologies as diagnostic and/or therapeutic reagents. Whole antibodiesare large protein structures exemplified by the heterotetramericstructure of the IgG isotype, containing two light and two heavy chains.Such large molecules are sterically hindered in certain applications.For example, in treatments of solid tumors, whole antibodies do notreadily penetrate the interior of the tumor. Moreover, the relativelylarge size of whole antibodies presents a challenge to ensure that thein vivo administration of such molecules does not induce an immuneresponse. Further, generation of active antibody molecules typicallyinvolves the culturing of recombinant eukaryotic cells capable ofproviding appropriate post-translational processing of the nascentantibody molecules, and such cells can be difficult to culture anddifficult to induce in a manner that provides commercially useful yieldsof active antibody.

Recently, smaller immunoglobulin molecules have been constructed toovercome problems associated with whole immunoglobulin methodologies. Asingle-chain variable antibody fragment (scFv) comprises an antibodyheavy chain variable domain joined via a short peptide to an antibodylight chain variable domain (Huston et al., Proc. Natl. Acad. Sci. USA,1988, 85: 5879-83). Because of the small size of scFv molecules, theyexhibit more effective penetration into tissues than wholeimmunoglobulin. An anti-tumor scFv showed more rapid tumor penetrationand more even distribution through the tumor mass than the correspondingchimeric antibody (Yokota et al., Cancer Res. 1992, 52:3402-08).

Despite the advantages that scFv molecules bring to serotherapy, severaldrawbacks to this therapeutic approach exist. An scFv is rapidly clearedfrom the circulation, which may reduce toxic effects in normal cells,but such rapid clearance impedes delivery of a minimum effective dose tothe target tissue. Manufacturing adequate amounts of scFv foradministration to patients has been challenging due to difficulties inexpression and isolation of scFv that adversely affect the yield. Duringexpression, scFv molecules lack stability and often aggregate due topairing of variable regions from different molecules. Furthermore,production levels of scFv molecules in mammalian expression systems arelow, limiting the potential for efficient manufacturing of scFvmolecules for therapy (Davis et al, J Biol. Chem. 1990, 265:10410-18);Traunecker et al., EMBO J 1991, 10: 3655-59). Strategies for improvingproduction have been explored, including addition of glycosylation sitesto the variable regions (Jost, C. R. U.S. Pat. No. 5,888,773, Jost etal, J. Biol. Chem. 1994, 69: 26267-73).

Another disadvantage to using scFv for therapy is the lack of effectorfunction. An scFv without a cytolytic function, such as theantibody-dependent cell-mediated cytotoxicity (ADCC) and complementdependent-cytotoxicity (CDC) associated with the constant region of animmunoglobulin, may be ineffective for treating disease. Even thoughdevelopment of scFv technology began over 12 years ago, currently noscFv products are approved for therapy.

Alternatively, it has been proposed that fusion of an scFv to anothermolecule, such as a toxin, could take advantage of the specificantigen-binding activity and the small size of an scFv to deliver thetoxin to a target tissue. Chaudary et al., Nature 1989, 339:394; Batraet al., Mol. Cell. Biol. 1991, 11:2200. Conjugation or fusion of toxinsto scFvs has thus been offered as an alternative strategy to providepotent, antigen-specific molecules, but dosing with such conjugates orchimeras can be limited by excessive and/or non-specific toxicity due tothe toxin moiety of such preparations. Toxic effects may includesupraphysiological elevation of liver enzymes and vascular leaksyndrome, and other undesired effects. In addition, immunotoxins arethemselves highly immunogenic upon administration to a host, and hostantibodies generated against the immunotoxin limit potential usefulnessfor repeated therapeutic treatments of an individual.

Nonsurgical cancer therapy, such as external irradiation andchemotherapy, can suffer from limited efficacy because of toxic effectson normal tissues and cells, due to the lack of specificity thesetreatments exhibit towards cancer cells. To overcome this limitation,targeted treatment methodologies have been developed to increase thespecificity of the treatment for the cells and tissues in need thereof.An example of such a targeted methodology for in vivo use is theadministration of antibody conjugates, with the antibody designed tospecifically recognize a marker associated with a cell or tissue in needof treatment, and the antibody being conjugated to a therapeutic agent,such as a toxin in the case of cancer treatment. Antibodies, as systemicagents, circulate to sensitive and undesirable body compartments, suchas the bone marrow. In acute radiation injury, destruction of lymphoidand hematopoietic compartments is a major factor in the development ofsepticemia and subsequent death. Moreover, antibodies are large,globular proteins that can exhibit poor penetration of tissues in needof treatment.

Human patients and non-human subjects suffering from a variety ofend-stage disease processes frequently require organ transplantation.Organ transplantation, however, must contend with the untoward immuneresponse of the recipient and guard against immunological rejection ofthe transplanted organ by depressing the recipient's cellular immuneresponse to the foreign organ with cytotoxic agents which affect thelymphoid and other parts of the hematopoietic system. Graft acceptanceis limited by the tolerance of the recipient to these cytotoxicchemicals, many of which are similar to the anticancer(antiproliferative) agents. Likewise, when using cytotoxic antimicrobialagents, particularly antiviral drugs, or when using cytotoxic drugs forautoimmune disease therapy, e.g., in treatment of systemic lupuserythematosis, a serious limitation is the toxic effects of thetherapeutic agents on the bone marrow and the hematopoietic cells of thebody.

Use of targeted therapies, such as targeted antibody conjugate therapy,is designed to localize a maximum quantity of the therapeutic agent atthe site of desired action as possible, and the success of suchtherapies is revealed by the relatively high signal-to-background ratioof therapeutic agent. Examples of targeted antibodies include diagnosticor therapeutic agent conjugates of antibody or antibody fragments, cell-or tissue-specific peptides, and hormones and other receptor-bindingmolecules. For example, antibodies against different determinantsassociated with pathological and normal cells, as well as associatedwith pathogenic microorganisms, have been used for the detection andtreatment of a wide variety of pathological conditions or lesions. Inthese methods, the targeting antibody is directly conjugated to anappropriate detecting or therapeutic agent as described, for example, inHansen et al., U.S. Pat. No. 3,927,193 and Goldenberg, U.S. Pat. Nos.4,331,647, 4,348,376, 4,361,544, 4,468,457, 4,444,744, 4,460,459,4,460,561, 4,624,846 and 4,818,709.

One problem encountered in direct targeting methods, i.e., in methodswherein the diagnostic or therapeutic agent (the “active agent”) isconjugated directly to the targeting moiety, is that a relatively smallfraction of the conjugate actually binds to the target site, while themajority of conjugate remains in circulation and compromises in one wayor another the function of the targeted conjugate. To ensure maximallocalization of the active agent, an excess of the targeted conjugate istypically administered, ensuring that some conjugate will remain unboundand contribute to background levels of the active agent. A diagnosticconjugate, e.g., a radioimmunoscintigraphic or magnetic resonanceimaging conjugate that does not bind its target can remain incirculation, thereby increasing background and decreasing resolution ofthe diagnostic technique. In the case of a therapeutic conjugate havinga toxin as an active agent (e.g., a radioisotope, drug or toxiccompound) attached to a long-circulating targeting moiety such as anantibody, circulating conjugate can result in unacceptable toxicity tothe host, such as marrow toxicity or systemic side effects.

U.S. Pat. No. 4,782,840 discloses a method for reducing the effect ofelevated background radiation levels during surgery. The method involvesinjection of a patient with antibodies specific for neoplastic tissue,with the antibodies labeled with radioisotopes having a suitably longhalf-life, such as Iodine-125. After injection of the radiolabeledantibody, the surgery is delayed at least 7-10 days, preferably 14-21days, to allow any unbound radiolabeled antibody to be cleared to a lowbackground level.

U.S. Pat. No. 4,932,412 discloses methods for reducing or correcting fornon-specific background radiation during intraoperative detection. Themethods include the administration to a patient who has received aradiolabeled primary antibody, of a contrast agent, subtraction agent orsecond antibody which binds the primary antibody.

Apart from producing the antibodies described above, the immune systemincludes a variety of cell types that have powerful biological effects.During hematopoiesis, bone marrow-derived stem cells differentiate intoeither mature cells of the immune system (“B” cells) or into precursorsof cells that migrate out of the bone marrow to mature in the thymus(“T” cells).

B cells are central to the humoral component of an immune response. Bcells are activated by an appropriate presentation of an antigen tobecome antibody-secreting plasma cells; antigen presentation alsoresults in clonal expansion of the activated B cell. B cells areprimarily responsible for the humoral component of an immune response. Aplasma cell typically exhibits about 10⁵ antibody molecules (IgD andIgM) on its surface.

T lymphocytes can be divided into two categories. The cytotoxic T cells,Tc lymphocytes or CTLs (CD8+ T cells), kill cells bearing foreignsurface antigen in association with Class I MHC and can kill cells thatare harboring intracellular parasites (either bacteria or viruses) aslong as the infected cell is displaying a microbial antigen on itssurface. Tc cells kill tumor cells and account for the rejection oftransplanted cells. Tc cells recognize antigen-Class I MHC complexes ontarget cells, contact them, and release the contents of granulesdirectly into the target cell membrane, which lyses the cell.

A second category of T cells is the helper T cell or Th lymphocyte (CD4+T cells), which produces lymphokines that are “helper” factors in thematuration of B cells into antibody-secreting plasma cells. Th cellsalso produce certain lymphokines that stimulate the differentiation ofeffector T lymphocytes and the activity of macrophages. Th1 cellsrecognize antigen on macrophages in association with Class II MHC andbecome activated (by IL-1) to produce lymphokines, including the IFN-γthat activates macrophages and NK cells. These cells mediate variousaspects of the cell-mediated immunity response including delayed-typehypersensitivity reactions. Th2 cells recognize antigen in associationwith Class II MHC on an antigen presenting cell or APC (e.g., migratorymacrophages and dendritic cells) and then produce interleukins and othersubstances that stimulate specific B-cell and T-cell proliferation andactivity.

Beyond serving as APCs that initiate T cell interactions, development,and proliferation, macrophages are involved in expression ofcell-mediated immunity because they become activated by IFN-γ producedin a cell-mediated immune response. Activated macrophages have increasedphagocytic potential and release soluble substances that causeinflammation and destroy many bacteria and other cells. Natural Killercells are cytotoxic cells that lyse cells bearing new antigen,regardless of their MHC type, and even lyse some cells that bear no MHCproteins. Natural Killer T cells, or NK cells, are defined by theirability to kill cells displaying a foreign antigen (e.g., tumor cells),regardless of MHC type, and regardless of previous sensitization(exposure) to the antigen. NK cells can be activated by IL-2 and IFN-γ,and lyse cells in the same manner as cytotoxic T lymphocytes. Some NKcells have receptors for the Fc domain of the IgG antibody (e.g, CD16 orF_(C)γRIII) and are thus able to bind to the Fc portion of IgG on thesurface of a target cell and release cytolytic components that kill thetarget cell via antibody-dependent cell-mediated cytotoxicity.

Another group of cells is the granulocytes or polymorphonuclearleukocytes (PMNs). Neutrophils, one type of PMN, kill bacterial invadersand phagocytose the remains. Eosinophils are another type of PMN andcontain granules that prove cytotoxic when released upon another cell,such as a foreign cell. Basophils, a third type of PMN, are significantmediators of powerful physiological responses (e.g., inflammation) thatexert their effects by releasing a variety of biologically activecompounds, such as histamine, serotonin, prostaglandins, andleukotrienes. Common to all of these cell types is the capacity to exerta physiological effect within an organism, frequently by killing, andoptionally scavenging, deleterious compositions such as foreign cells.

Although a variety of mammalian cells, including cells of the immunesystem, are capable of directly exerting a physiological effect (e.g.,cell killing, typified by Tc, NK, some PMN, macrophage, and the like),other cells indirectly contribute to a physiological effect. Forexample, initial presentation of an antigen to a naïve T cell of theimmune system requires MHC presentation that mandates cell-cell contact.Further, there often needs to be contact between an activated T cell andan antigen-specific B cell to obtain a particular immunogenic response.A third form of cell-cell contact often seen in immune responses is thecontact between an activated B cell and follicular dendritic cells. Eachof these cell-cell contact requirements complicates the targeting of abiologically active agent to a given target.

Complement-dependent cytotoxicity (CDC) is believed to be a significantmechanism for clearance of specific target cells such as tumor cells.CDC is a series of events that consists of a collection of enzymes thatbecome activated by each other in a cascade fashion. Complement has animportant role in clearing antigen, accomplished by its four majorfunctions: (1) local vasodilation; (2) attraction of immune cells,especially phagocytes (chemotaxis); (3) tagging of foreign organisms forphagocytosis (opsonization); and (4) destruction of invading organismsby the membrane attack complex (MAC attack). The central molecule is theC3 protein. It is an enzyme that is split into two fragments bycomponents of either the classical pathway or the alternative pathway.The classical pathway is induced by antibodies, especially IgG and IgM,while the alternative pathway is nonspecifically stimulated by bacterialproducts like lipopolysaccharide (LPS). Briefly, the products of the C3split include a small peptide C3a which is chemotactic for phagocyticimmune cells and results in local vasodilation by causing the release ofC5a fragment from C5. The other part of C3, C3b, coats antigens on thesurface of foreign organisms and acts to opsonize the organism fordestruction. C3b also reacts with other components of the complementsystem to form an MAC consisting of C5b, C6, C7, C8 and C9.

There are problems associated with the use of antibodies in humantherapy because the response of the immune system to any antigen, eventhe simplest, is “polyclonal,” i.e., the system manufactures antibodiesof a great range of structures both in their binding regions as well asin their effector regions.

Two approaches have been used in an attempt to reduce the problem ofimmunogenic antibodies. The first is the production of chimericantibodies in which the antigen-binding part (variable regions) of amouse monoclonal antibody is fused to the effector part (constantregion) of a human antibody. In a second approach, antibodies have beenaltered through a technique known as complementarity determining region(CDR) grafting or “humanization.” This process has been further improvedto include changes referred to as “reshaping” (Verhoeyen, et al., 1988Science 239:1534-1536; Riechmann, et al., 1988 Nature 332:323-337;Tempest, et al., Bio/Technol 1991 9:266-271), “hyperchimerization”(Queen, et al., 1989 Proc Natl Acad Sci USA 86:10029-10033; Co, et al.,1991 Proc Natl Acad Sci USA 88:2869-2873; Co, et al., 1992 J Immunol148:1149-1154), and “veneering” (Mark, et al., In: Metcalf B W, Dalton BJ, eds. Cellular adhesion: molecular definition to therapeuticpotential. New York: Plenum Press, 1994:291-312).

An average of less than one therapeutic antibody per year has beenintroduced to the market beginning in 1986, eleven years after thepublication of monoclonal antibodies. Five murine monoclonal antibodieswere introduced into human medicine over a ten year period from1986-1995, including “muromonab-CD3” (OrthoClone OKT3®) for acuterejection of organ transplants; “edrecolomab” (Panorex®) for colorectalcancer; “odulimomab” (Antilfa®) or transplant rejection; and,“ibritumomab” (Zevalin® yiuxetan) for non-Hodgkin's lymphoma.Additionally, a monoclonal Fab, “abciximab” (ReoPro®) has been marketedfor preventing coronary artery reocclusion. Three chimeric monoclonalantibodies were also launched: “rituximab” (Rituxan®) for treating Bcell lymphomas; “basiliximab” (Simulect®) for transplant rejection; and“infliximab” (Remicade®) for treatment of rheumatoid arthritis andCrohn's disease. Additionally, “abciximab” (ReoPro®), a 47.6 kD Fabfragment of a chimeric human-murine monoclonal antibody is marketed asan adjunct to percutaneous coronary intervention for the prevention ofcardiac ischemic complications in patients undergoing percutaneouscoronary intervention. Finally, seven “humanized” monoclonal antibodieshave been launched. “Daclizumab” (Zenapax®) is used to prevent acuterejection of transplanted kidneys; “palivizumab” (Synagis®) for RSV;“trastuzumab” (Herceptin®) binds HER-2, a growth factor receptor foundon breast cancers cells; “gemtuzumab” (Mylotarg®) for acute myelogenousleukemia (AML); and “alemtuzumab” (MabCampath®) for chronic lymphocyticleukemia; “adalimumab” (Humira® (D2E7)) for the treatment of rheumatoidarthritis; and, “omalizumab” (Xolair®), for the treatment of persistentasthma.

Thus, a variety of antibody technologies have received attention in theeffort to develop and market more effective therapeutics andpalliatives. Unfortunately, problems continue to compromise the promiseof each of these therapies. For example, the majority of cancer patientstreated with rituximab relapse, generally within about 6-12 months, andfatal infusion reactions within 24 hours of rituximab infusion have beenreported. Acute renal failure requiring dialysis with instances of fataloutcome has also been reported in treatments with rituximab, as havesevere, occasionally fatal, mucocutaneous reactions. Additionally, highdoses of rituximab are required for intravenous injection because themolecule is large, approximately 150 kDa, and diffusion into thelymphoid tissues, where many tumor cells may reside is limited.

Trastuzumab administration can result in the development of ventriculardysfunction, congestive heart failure, and severe hypersensitivityreactions (including anaphylaxis), infusion reactions, and pulmonaryevents. Daclizumab immunosuppressive therapy poses an increased risk fordeveloping lymphoproliferative disorders and opportunistic infections.Death from liver failure, arising from severe hepatotoxicity, and fromveno-occlusive disease (VOD), has been reported in patients who receivedgemtuzumab.

Hepatotoxicity was also reported in patients receiving alemtuzumab.Serious and, in some rare instances fatal, pancytopenia/marrowhypoplasia, autoimmune idiopathic thrombocytopenia, and autoimmunehemolytic anemia have occurred in patients receiving alemtuzumabtherapy. Alemtuzumab can also result in serious infusion reactions aswell as opportunistic infections. In patients treated with adalimumab,serious infections and sepsis, including fatalities, have been reported,as has the exacerbation of clinical symptoms and/or radiographicevidence of demyelinating disease, and patients treated with adalimumabin clinical trials had a higher incidence of lymphoma than the expectedrate in the general population. Omalizumab reportedly inducesmalignancies and anaphylaxis.

Cancer includes a broad range of diseases, affecting approximately onein four individuals worldwide. Rapid and unregulated proliferation ofmalignant cells is a hallmark of many types of cancer, includinghematological malignancies. Although patients with a hematologicmalignant condition have benefited from advances in cancer therapy inthe past two decades, Multani et al., 1998 J. Clin. Oncology16:3691-3710, and remission times have increased, most patients stillrelapse and succumb to their disease. Barriers to cure with cytotoxicdrugs include, for example, tumor cell resistance and the high toxicityof chemotherapy, which prevents optimal dosing in many patients.

Treatment of patients with low grade or follicular B cell lymphoma usinga chimeric CD20 monoclonal antibody has been reported to induce partialor complete responses in patients. McLaughlin et al., 1996 Blood 88:90a(abstract, suppl. 1); Maloney et al., 1997 Blood 90:2188-95. However, asnoted above, tumor relapse commonly occurs within six months to oneyear. Further improvements in serotherapy are needed to induce moredurable responses, for example, in low grade B cell lymphoma, and toallow effective treatment of high grade lymphoma and other B celldiseases.

Another approach has been to target radioisotopes to B cell lymphomasusing monoclonal antibodies specific for CD20. While the effectivenessof therapy is reportedly increased, associated toxicity from the long invivo half-life of the radioactive antibody increases, sometimesrequiring that the patient undergo stem cell rescue. Press et al., 1993N. Eng. J. Med. 329:1219-1224; Kaminski et al., 1993 N. Eng. J. Med.329:459-65. Monoclonal antibodies to CD20 have also been cleaved withproteases to yield F(ab′)₂ or Fab fragments prior to attachment ofradioisotope. This has been reported to improve penetration of theradioisotope conjugate into the tumor and to shorten the in vivohalf-life, thus reducing the toxicity to normal tissues. However, thesemolecules lack effector functions, including complement fixation and/orADCC.

Autoimmune diseases include autoimmune thyroid diseases, which includeGraves' disease and Hashimoto's thyroiditis. In the United States alone,there are about 20 million people who have some form of autoimmunethyroid disease. Autoimmune thyroid disease results from the productionof autoantibodies that either stimulate the thyroid to causehyperthyroidism (Graves' disease) or destroy the thyroid to causehypothyroidism (Hashimoto's thyroiditis). Stimulation of the thyroid iscaused by autoantibodies that bind and activate the thyroid stimulatinghormone (TSH) receptor. Destruction of the thyroid is caused byautoantibodies that react with other thyroid antigens. Current therapyfor Graves' disease includes surgery, radioactive iodine, or antithyroiddrug therapy. Radioactive iodine is widely used, since antithyroidmedications have significant side effects and disease recurrence ishigh. Surgery is reserved for patients with large goiters or where thereis a need for very rapid normalization of thyroid function. There are notherapies that target the production of autoantibodies responsible forstimulating the TSH receptor. Current therapy for Hashimoto'sthyroiditis is levothyroxine sodium, and lifetime therapy is expectedbecause of the low likelihood of remission. Suppressive therapy has beenshown to shrink goiters in Hashimoto's thyroiditis, but no therapiesthat reduce autoantibody production to target the disease mechanism areknown.

Rheumatoid arthritis (RA) is a chronic disease characterized byinflammation of the joints, leading to swelling, pain, and loss offunction. RA affects an estimated 2.5 million people in the UnitedStates. RA is caused by a combination of events including an initialinfection or injury, an abnormal immune response, and genetic factors.While autoreactive T cells and B cells are present in RA, the detectionof high levels of antibodies that collect in the joints, calledrheumatoid factor, is used in the diagnosis of RA. Current therapy forRA includes many medications for managing pain and slowing theprogression of the disease. No therapy has been found that can cure thedisease. Medications include nonsteroidal anti-inflammatory drugs(NSAIDS), and disease modifying anti-rheumatic drugs (DMARDS). NSAIDSare useful in benign disease, but fail to prevent the progression tojoint destruction and debility in severe RA. Both NSA/DS and DMARDS areassociated with significant side effects. Only one new DMARD,Leflunomide, has been approved in over 10 years. Leflunomide blocksproduction of autoantibodies, reduces inflammation, and slowsprogression of RA. However, this drug also causes severe side effectsincluding nausea, diarrhea, hair loss, rash, and liver injury.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease caused byrecurrent injuries to blood vessels in multiple organs, including thekidney, skin, and joints. SLE is estimated to affect over 500,000 peoplein the United States. In patients with SLE, a faulty interaction betweenT cells and B cells results in the production of autoantibodies thatattack the cell nucleus. These include anti-double stranded DNA andanti-Sm antibodies. Autoantibodies that bind phospholipids are alsofound in about half of SLE patients, and are responsible for bloodvessel damage and low blood counts. Immune complexes accumulate in thekidneys, blood vessels, and joints of SLE patients, where they causeinflammation and tissue damage. No treatment for SLE has been found tocure the disease. NSAIDS and DMARDS are used for therapy depending uponthe severity of the disease. Plasmapheresis with plasma exchange toremove autoantibodies can cause temporary improvement in SLE patients.There is general agreement that autoantibodies are responsible for SLE,so new therapies that deplete the B cell lineage, allowing the immunesystem to reset as new B cells are generated from precursors, wouldoffer hope for long lasting benefit in SLE patients.

Sjogren's syndrome is an autoimmune disease characterized by destructionof the body's moisture-producing glands. Sjogren's syndrome is one ofthe most prevalent autoimmune disorders, striking up to an estimated 4million people in the United States. About half of the people strickenwith Sjogren's syndrome also have a connective tissue disease, such asRA, while the other half have primary Sjogren's syndrome with no otherconcurrent autoimmune disease. Autoantibodies, including anti-nuclearantibodies, rheumatoid factor, anti-fodrin, and anti-muscarinic receptorare often present in patients with Sjogren's syndrome. Conventionaltherapy includes corticosteroids, and additional more effectivetherapies would be of benefit.

Immune thrombocytopenic purpura (ITP) is caused by autoantibodies thatbind to blood platelets and cause their destruction. Some cases of ITPare caused by drugs, and others are associated with infection,pregnancy, or autoimmune disease such as SLE. About half of all casesare classified as being of idiopathic origin. The treatment of ITP isdetermined by the severity of the symptoms. In some cases, no therapy isneeded although in most cases immunosuppressive drugs, includingcorticosteroids or intravenous infusions of immune globulin to deplete Tcells, are provided. Another treatment that usually results in anincreased number of platelets is removal of the spleen, the organ thatdestroys antibody-coated platelets. More potent immunosuppressive drugs,including cyclosporine, cyclophosphamide, or azathioprine are used forpatients with severe cases. Removal of autoantibodies by passage ofpatients' plasma over a Protein A column is used as a second linetreatment in patients with severe disease. Additional more effectivetherapies are needed.

Multiple sclerosis (MS) is also an autoimmune disease. It ischaracterized by inflammation of the central nervous system anddestruction of myelin, which insulates nerve cell fibers in the brain,spinal cord, and body. Although the cause of MS is unknown, it is widelybelieved that autoimmune T cells are primary contributors to thepathogenesis of the disease. However, high levels of antibodies arepresent in the cerebrospinal fluid of patients with MS, and some predictthat the B cell response leading to antibody production is important formediating the disease. No B cell depletion therapies have been studiedin patients with MS, and there is no cure for MS. Current therapy iscorticosteroids, which can reduce the duration and severity of attacks,but do not affect the course of MS over time. New biotechnologyinterferon (IFN) therapies for MS have recently been approved butadditional more effective therapies are required.

Myasthenia Gravis (MG) is a chronic autoimmune neuromuscular disorderthat is characterized by weakness of the voluntary muscle groups. MGaffects about 40,000 people in the United States. MG is caused byautoantibodies that bind to acetylcholine receptors expressed atneuromuscular junctions. The autoantibodies reduce or blockacetylcholine receptors, preventing the transmission of signals fromnerves to muscles. There is no known cure for mg. Common treatmentsinclude immunosuppression with corticosteroids, cyclosporine,cyclophosphamide, or azathioprine. Surgical removal of the thymus isoften used to blunt the autoimmune response. Plasmapheresis, used toreduce autoantibody levels in the blood, is effective in mg, but isshort-lived because the production of autoantibodies continues.Plasmapheresis is usually reserved for severe muscle weakness prior tosurgery. New and effective therapies would be of benefit.

Psoriasis affects approximately five million people, and ischaracterized by autoimmune inflammation in the skin. Psoriasis is alsoassociated with arthritis in 30% (psoriatic arthritis). Many treatments,including steroids, uv light retinoids, vitamin D derivatives,cyclosporine, and methotrexate have been used but it is also clear thatpsoriasis would benefit from new and effective therapies. Scleroderma isa chronic autoimmune disease of the connective tissue that is also knownas systemic sclerosis. Scleroderma is characterized by an overproductionof collagen, resulting in a thickening of the skin, and approximately300,000 people in the United States have scleroderma, which would alsobenefit from new and effective therapies.

Apparent from the foregoing discussion are needs for improvedcompositions and methods to treat, ameliorate or prevent a variety ofdiseases, disorders and conditions, including cancer and autoimmunediseases.

SUMMARY

The invention satisfies at least one of the aforementioned needs in theart by providing proteins containing at least two specific bindingdomains, wherein those two domains are linked by a constant sub-regionderived from an antibody molecule attached at its C-terminus to a linkerherein referred to as a scorpion linker, and nucleic acids encoding suchproteins, as well as production, diagnostic and therapeutic uses of suchproteins and nucleic acids. The constant sub-region comprises a domainderived from an immunoglobulin C_(H2) domain, and preferably a domainderived from an immunoglobulin C_(H3) domain, but does not contain adomain or region derived from, or corresponding to, an immunoglobulinC_(H1) domain. Previously, it had been thought that the placement of aconstant region derived from an antibody in the interior of a proteinwould interfere with antibody function, such as effector function, byanalogy to the conventional placement of constant regions of antibodiesat the carboxy termini of antibody chains. In addition, placement of ascorpion linker, which may be an immunoglobulin hinge-like peptide,C-terminal to a constant sub-region is an organization that differs fromthe organization of naturally occurring immunoglobulins. Placement of aconstant sub-region (with a scorpion linker attached C-terminal to theconstant region) in the interior of a polypeptide or protein chain inaccordance with the invention, however, resulted in proteins exhibitingeffector function and multivalent (mono- or multi-specific) bindingcapacities relatively unencumbered by steric hindrances. As will beapparent to one of skill in the art upon consideration of thisdisclosure, such proteins are modular in design and may be constructedby selecting any of a variety of binding domains for binding domain 1 orbinding domain 2 (or for any additional binding domains found in aparticular protein according to the invention), by selecting a constantsub-region having effector function, and by selecting a scorpion linker,hinge-like or non-hinge like (e.g., type II C-lectin receptor stalkregion peptides), with the protein exhibiting a general organization ofN-binding domain 1-constant sub-region-scorpion linker-binding domain2-C. Those of skill will further appreciate that proteins of suchstructure, and the nucleic acids encoding those proteins, will find awide variety of applications, including medical and veterinaryapplications.

One aspect of the invention is drawn to a multivalent single-chainbinding protein with effector function, or scorpion (the terms are usedinterchangeably), comprising a first binding domain derived from animmunoglobulin (e.g., an antibody) or an immunoglobulin-like molecule, aconstant sub-region providing an effector function, the constantsub-region located C-terminal to the first binding domain; a scorpionlinker located C-terminal to the constant sub-region; and a secondbinding domain derived from an immunoglobulin (such as an antibody) orimmunoglobulin-like molecule, located C-terminal to the constantsub-region; thereby localizing the constant sub-region between the firstbinding domain and the second binding domain. The single-chain bindingprotein may be multispecific, e.g., bispecific in that it could bind twoor more distinct targets, or it may be monospecific, with two bindingsites for the same target. Moreover, all of the domains of the proteinare found in a single chain, but the protein may form homo-multimers,e.g., by interchain disulfide bond formation. In some embodiments, thefirst binding domain and/or the second binding domain is/are derivedfrom variable regions of light and heavy immunoglobulin chains from thesame, or different, immunoglobulins (e.g., antibodies). Theimmunoglobulin(s) may be from any vertebrate, such as a mammal,including a human, and may be chimeric, humanized, fragments, variantsor derivatives of naturally occurring immunoglobulins.

The invention contemplates proteins in which the first and secondbinding domains are derived from the same, or different immunoglobulins(e.g., antibodies), and wherein the first and second binding domainsrecognize the same, or different, molecular targets (e.g., cell surfacemarkers, such as membrane-bound proteins). Further, the first and secondbinding domains may recognize the same, or different, epitopes. Thefirst and second molecular targets may be associated with first andsecond target cells, viruses, carriers and/or objects. In preferredembodiments according to this aspect of the invention, each of the firstbinding domain, second binding domain, and constant sub-region isderived from a human immunoglobulin, such as an IgG antibody. In yetother embodiments, the multivalent binding protein with effectorfunction has at least one of the first binding domain and the secondbinding domain that recognizes at least one cell-free molecular target,e.g., a protein unassociated with a cell, such as a deposited protein ora soluble protein. Cell-free molecular targets include, e.g., proteinsthat were never associated with a cell, e.g., administered compoundssuch as proteins, as well as proteins that are secreted, cleaved,present in exosomes, or otherwise discharged or separated from a cell.

The target molecules recognized by the first and second binding domainsmay be found on, or in association with, the same, or different,prokaryotic cells, eukaryotic cells, viruses (including bacteriophage),organic or inorganic target molecule carriers, and foreign objects.Moreover, those target molecules may be on physically distinct cells,viruses, carriers or objects of the same type (e.g., two distincteukaryotic cells, prokaryotic cells, viruses or carriers) or thosetarget molecules may be on cells, viruses, carriers, or objects thatdiffer in type (e.g., a eukaryotic cell and a virus). Target cells arethose cells associated with a target molecule recognized by a bindingdomain and includes endogenous or autologous cells as well as exogenousor foreign cells (e.g., infectious microbial cells, transplantedmammalian cells including transfused blood cells). The inventioncomprehends targets for the first and/or second binding domains that arefound on the surface of a target cell(s) associated with a disease,disorder or condition of a mammal such as a human. Exemplary targetcells include a cancer cell, a cell associated with an autoimmunedisease or disorder, and an infectious cell (e.g., an infectiousbacterium). A cell of an infectious organism, such as a mammalianparasite, is also contemplated as a target cell. In some embodiments, aprotein of the invention is a multivalent (e.g., multispecific) bindingprotein with effector function wherein at least one of the first bindingdomain and the second binding domain recognizes a target selected fromthe group consisting of a tumor antigen, a B-cell target, a TNF receptorsuperfamily member, a Hedgehog family member, a receptor tyrosinekinase, a proteoglycan-related molecule, a TGF-beta superfamily member,a Wnt-related molecule, a receptor ligand, a T-cell target, a Dendriticcell target, an NK cell target, a monocyte/macrophage cell target and anangiogenesis target.

In some embodiments of the above-described protein, the tumor antigen isselected from the group consisting of SQUAMOUS CELL CARCINOMA ANTIGEN 1(SCCA-1), (PROTEIN T4-A), SQUAMOUS CELL CARCINOMA ANTIGEN 2 (SCCA-2),Ovarian carcinoma antigen CA125 (1A1-3B) (KIAA0049), MUCIN 1(TUMOR-ASSOCIATED MUCIN), (CARCINOMA-ASSOCIATED MUCIN), (POLYMORPHICEPITHELIAL MUCIN), (PEM), (PEMT), (EPISIALIN), (TUMOR-ASSOCIATEDEPITHELIAL MEMBRANE ANTIGEN), (EMA), (H23AG), (PEANUT-REACTIVE URINARYMUCIN), (PUM), (BREAST CARCINOMA-ASSOCIATED ANTIGEN DF3), CTCL tumorantigen se1-1, CTCL tumor antigen se14-3, CTCL tumor antigen se20-4,CTCL tumor antigen se20-9, CTCL tumor antigen se33-1, CTCL tumor antigense37-2, CTCL tumor antigen se57-1, CTCL tumor antigen se89-1,Prostate-specific membrane antigen, 5T4 oncofetal trophoblastglycoprotein, Orf73 Kaposi's sarcoma-associated herpesvirus, MAGE-C1(cancer/testis antigen CT7), MAGE-BI ANTIGEN (MAGE-XP ANTIGEN) (DAM10),MAGE-B2 ANTIGEN (DAM6), MAGE-2 ANTIGEN, MAGE-4a antigen, MAGE-4bantigen, Colon cancer antigen NY-CO-45, Lung cancer antigen NY-LU-12variant A, Cancer associated surface antigen, Adenocarcinoma antigenART1, Paraneoplastic associated brain-testis-cancer antigen(onconeuronal antigen MA2; paraneoplastic neuronal antigen),Neuro-oncological ventral antigen 2 (NOVA2), Hepatocellular carcinomaantigen gene 520, TUMOR-ASSOCIATED ANTIGEN CO-029, Tumor-associatedantigen MAGE-X2, Synovial sarcoma, X breakpoint 2, Squamous cellcarcinoma antigen recognized by T cell, Serologically defined coloncancer antigen 1, Serologically defined breast cancer antigen NY-BR-15,Serologically defined breast cancer antigen NY-BR-16, Chromogranin A;parathyroid secretory protein 1, DUPAN-2, CA 19-9, CA 72-4, CA 195 andL6.

Embodiments of the above-described method comprise a B cell targetselected from the group consisting of CD10, CD19, CD20, CD21, CD22,CD23, CD24, CD37, CD38, CD39, CD40, CD72, CD73, CD74, CDw75, CDw76,CD77, CD78, CD79a/b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD89,CD98, CD126, CD127, CDw130, CD138 and CDw150.

In other embodiments of the above-described method, the TNF receptorsuperfamily member is selected from the group consisting of4-1BB/TNFRSF9, NGF R/TNFRSF16, BAFF R/TNFRSF13C,Osteoprotegerin/TNFRSF11B, BCMA/TNFRSF17, OX40/TNFRSF4, CD27/TNFRSF7,RANK/TNFRSF11A, CD30/TNFRSF8, RELT/TNFRSF19L, CD40/TNFRSF5,TACI/TNFRSF13B, DcR3/TNFRSF6B, TNF RI/TNFRSF1A, DcTRAIL R1/TNFRSF23, TNFRII/TNFRSF1B, DcTRAIL R2/TNFRSF22, TRAIL R1/INFRSF10A, DR3/TNFRSF25,TRAIL R2/TNFRSF10B, DR6/TNFRSF21, TRAIL R3/TNFRSF10C, EDAR, TRAILR4/TNFRSF10D, Fas/TNFRSF6, TROY/TNFRSF19, GITR/TNFRSF18, TWEAKR/TNFRSF12, HVEM/TNFRSF14, XEDAR, Lymphotoxin beta R/TNFRSF3, 4-1BBLigand/TNFSF9, Lymphotoxin, APRIL/TNFSF13, Lymphotoxin beta/TNFSF3,BAFF/TNFSF13C, OX40 Ligand/TNFSF4, CD27 Ligand/TNFSF7, TL1A/TNFSF15,CD30 Ligand/TNFSF8, TNF-alpha/TNFSF1A, CD40 Ligand/TNFSF5,TNF-beta/TNFSF1B, EDA-A2, TRAIL/TNFSF10, Fas Ligand/TNFSF6,TRANCE/TNFSF11, GITR Ligand/TNFSF18, TWEAK/TNFSF12 and LIGHT/TNFSF14.

The above-described method also includes embodiments in which theHedgehog family member is selected from the group consisting of Patchedand Smoothened. In yet other embodiments, the proteoglycan-relatedmolecule is selected from the group consisting of proteoglycans andregulators thereof.

Additional embodiments of the method are drawn to processes in which thereceptor tyrosine kinase is selected from the group consisting of Axl,FGF R4, C1q R1/CD93, FGF R5, DDR1, Flt-3, DDR2, HGF R, Dtk, IGF-I R, EGFR, IGF-II R, Eph, INSRR, EphA1, Insulin R/CD220, EphA2, M-CSF R, EphA3,Mer, EphA4, MSP R/Ron, EphA5, MuSK, EphA6, PDGF R alpha, EphA7, PDGF Rbeta, EphA8, Ret, EphB1, ROR1, EphB2, ROR2, EphB3, SCF R/c-kit, EphB4,Tie-1, EphB6, Tie-2, ErbB2, TrkA, ErbB3, TrkB, ErbB4, TrkC, FGF R1, VEGFR1/Flt-I, FGF R2, VEGF R2/Flk-1, FGF R3 and VEGF R3/Flt-4.

In other embodiments of the method, the Transforming Growth Factor(TGF)-beta superfamily member is selected from the group consisting ofActivin RIA/ALK-2, GFR alpha-1, Activin RIB/ALK-4, GFR alpha-2, ActivinRIIA, GFR alpha-3, Activin RIIB, GFR alpha-4, ALK-1, MIS RII, ALK-7,Ret, BMPR-IA/ALK-3, TGF-beta RI/ALK-5, BMPR-IB/ALK-6, TGF-beta RII,BMPR-II, TGF-beta Endoglin/CD105 and TGF-beta RIII.

Yet other embodiments of the method comprise a Wnt-related moleculeselected from the group consisting of Frizzled-1, Frizzled-8,Frizzled-2, Frizzled-9, Frizzled-3, sFRP-1, Frizzled-4, sFRP-2,Frizzled-5, sFRP-3, Frizzled-6, sFRP-4, Frizzled-7, MFRP, LRP 5, LRP 6,Wnt-1, Wnt-8a, Wnt-3a, Wnt-10b, Wnt-4, Wnt-11, Wnt-5a, Wnt-9a andWnt-7a.

In other embodiments of the method, the receptor ligand is selected fromthe group consisting of 4-1BB Ligand/TNFSF9, Lymphotoxin, APRIL/TNFSF13,Lymphotoxin beta/TNFSF3, BAFF/TNFSF13C, OX40 Ligand/TNFSF4, CD27Ligand/TNFSF7, TL1A/TNFSF15, CD30 Ligand/TNFSF8, TNF-alpha/TNFSF1A, CD40Ligand/TNFSF5, TNF-beta/TNFSF1B, EDA-A2, TRAIL/TNFSF10, FasLigand/TNFSF6, TRANCE/TNFSF11, GITR Ligand/TNFSF18, TWEAK/TNFSF12,LIGHT/TNFSF14, Amphiregulin, NRG1 isoform GGF2, Betacellulin, NRG1Isoform SMDF, EGF, NRG1-alpha/HRG1-alpha, Epigen, NRG1-beta 1/HRG1-beta1, Epiregulin, TGF-alpha, HB-EGF, TMEFF1/Tomoregulin-1, Neuregulin-3,TMEFF2, IGF-I, IGF-II, Insulin, Activin A, Activin B, Activin AB,Activin C, BMP-2, BMP-7, BMP-3, BMP-8, BMP-3b/GDF-10, BMP-9, BMP-4,BMP-15, BMP-5, Decapentaplegic, BMP-6, GDF-1, GDF-8, GDF-3, GDF-9,GDF-5, GDF-11, GDF-6, GDF-15, GDF-7, Artemin, Neurturin, GDNF,Persephin, TGF-beta, TGF-beta 2, TGF-beta 1, TGF-beta 3, LAP (TGF-beta1), TGF-beta 5, Latent TGF-beta 1, Latent TGF-beta bp1, TGF-beta 1.2,Lefty, Nodal, MIS/AMH, FGF acidic, FGF-12, FGF basic, FGF-13, FGF-3,FGF-16, FGF-4, FGF-17, FGF-5, FGF-19, FGF-6, FGF-20, FGF-8, FGF-21,FGF-9, FGF-23, FGF-10, KGF/FGF-7, FGF-11, Neuropilin-1, P1GF,Neuropilin-2, P1GF-2, PDGF, PDGF-A, VEGF, PDGF-B, VEGF-B, PDGF-C,VEGF-C, PDGF-D, VEGF-D and PDGF-AB.

In still other embodiments, the T-cell target is selected from the groupconsisting of 2B4/SLAMF4, IL-2 R alpha, 4-1BB/TNFRSF9, IL-2 R beta,ALCAM, B7-1/CD80, IL-4 R, B7-H3, BLAME/SLAMF8, BTLA, IL-6 R, CCR3, IL-7R alpha, CCR4, CXCR1/IL-8 RA, CCR5, CCR6, IL-10 R alpha, CCR7, IL-10 Rbeta, CCR8, IL-12 R beta 1, CCR9, IL-12 R beta 2, CD2, IL-13 R alpha 1,IL-13, CD3, CD4, ILT2/CD85j, ILT3/CD85k, ILT4/CD85d, ILT5/CD85a,Integrin alpha 4/CD49d, CD5, Integrin alpha E/CD103, CD6, Integrin alphaM/CD11b, CD8, Integrin alpha X/CD11c, Integrin beta 2/CD18, K1R/CD158,CD27/TNFRSF7, K1R2DL1, CD28, KIR2DL3, CD30/TNFRSF8, K1R2DL4/CD158d,CD31/PECAM-1, KIR2DS4, CD40 Ligand/TNFSF5, LAG-3, CD43, LAIR1, CD45,LAIR2, CD83, Leukotriene B4 R1, CD84/SLAMF5, NCAM-L1, CD94, NKG2A, CD97,NKG2C, CD229/SLAMF3, NKG2D, CD2F-10/SLAMF9, NT-4, CD69, NTB-A/SLAMF6,Common gamma Chain/IL-2 R gamma, Osteopontin, CRACC/SLAMF7, PD-1, CRTAM,PSGL-1, CTLA-4, RANK/TNFRSF11A, CX3CR1, CX3CL1, L-Selectin, CXCR3, SIRPbeta 1, CXCR4, SLAM, CXCR6, TCCR/WSX-1, DNAM-1, Thymopoietin,EMMPRIN/CD147, TIM-1, EphB6, TIM-2, Fas/TNFRSF6, TIM-3, FasLigand/TNFSF6, TIM-4, Fc gamma RIII/CD16, TIM-6, GITR/TNFRSF18, TNFRI/TNFRSF1A, Granulysin, TNF RII/TNFRSF1B, HVEM/TNFRSF14, TRAILR1/TNFRSF10A, ICAM-1/CD54, TRAIL R2/TNFRSF10B, ICAM-2/CD102, TRAILR3/TNFRSF10C, IFN-gamma R1, TRAIL R4/TNFRSF10D, IFN-gamma R2, TSLP, IL-1RI and TSLP R.

In other embodiments, the NK cell receptor is selected from the groupconsisting of 2B4/SLAMF4, KIR2DS4, CD155/PVR, KIR3DL1, CD94,LMIR1/CD300A, CD69, LMIR2/CD300c, CRACC/SLAMF7, LMIR3/CD300LF, DNAM-1,LMIR5/CD300LB, Fc epsilon RII, LMIR6/CD300LE, Fc gamma RI/CD64, MICA, Fcgamma RIIB/CD32b, MICB, Fc gamma RIIc/CD32c, MULT-1, Fc gammaRIIA/CD32a, Nectin-2/CD112, Fc gamma RIII/CD16, NKG2A, FcRH1/IRTA5,NKG2C, FcRH2/IRTA4, NKG2D, FcRH4/IRTA1, NKp30, FcRH5/IRTA2, NKp44, FcReceptor-like 3/CD16-2, NKp46/NCR1, NKp80/KLRF1, NTB-A/SLAMF6, Rae-1,Rae-1 alpha, Rae-1 beta, Rae-1 delta, H60, Rae-1 epsilon, ILT2/CD85j,Rae-1 gamma, ILT3/CD85k, TREM-1, ILT4/CD85d, TREM-2, ILT5/CD85a, TREM-3,KIR/CD158, TREML1/TLT-1, KIR2DL1, ULBP-1, KIR2DL3, ULBP-2,KIR2DL4/CD158d and ULBP-3.

In other embodiments, the monocyte/macrophage cell target is selectedfrom the group consisting of B7-1/CD80, ILT4/CD85d, B7-H1, ILT5/CD85a,Common beta Chain, Integrin alpha 4/CD49d, BLAME/SLAMF8, Integrin alphaX/CD11c, CCL6/C10, Integrin beta 2/CD18, CD155/PVR, Integrin beta3/CD61, CD31/PECAM-1, Latexin, CD36/SR-B3, Leukotriene B4 R1,CD40/TNFRSF5, LIMPII/SR-B2, CD43, LMIR1/CD300A, CD45, LMIR2/CD300c,CD68, LMIR3/CD300LF, CD84/SLAMF5, LMIR5/CD300LB, CD97, LMIR6/CD300LE,CD163, LRP-1, CD2F-10/SLAMF9, MARCO, CRACC/SLAMF7, MD-1, ECF-L, MD-2,EMMPRIN/CD147, MGL2, Endoglin/CD105, Osteoactivin/GPNMB, Fc gammaRI/CD64, Osteopontin, Fc gamma RIIB/CD32b, PD-L2, Fc gamma RIIC/CD32c,Siglec-3/CD33, Fc gamma RIIA/CD32a, SIGNR1/CD209, Fc gamma RIII/CD16,SLAM, GM-CSF R alpha, TCCR/WSX-1, ICAM-2/CD102, TLR3, IFN-gamma R1,TLR4, IFN-gamma R2, TREM-1, IL-1 RII, TREM-2, ILT2/CD85j, TREM-3,ILT3/CD85k, TREML1/TLT-1, 2B4/SLAMF4, IL-10 R alpha, ALCAM, IL-10 Rbeta, Aminopeptidase N/ANPEP, ILT2/CD85j, Common beta Chain, ILT3/CD85k,C1q R1/CD93, ILT4/CD85d, CCR1, ILT5/CD85a, CCR2, Integrin alpha 4/CD49d,CCR5, Integrin alpha M/CD11b, CCR8, Integrin alpha X/CD11c, CD155/PVR,Integrin beta 2/CD18, CD14, Integrin beta 3/CD61, CD36/SR-B3, LAIR1,CD43, LAIR2, CD45, Leukotriene B4 R1, CD68, LIMPII/SR-B2, CD84/SLAMF5,LMIR1/CD300A, CD97, LMIR2/CD300c, CD163, LMIR3/CD300LF, CoagulationFactor III/Tissue Factor, LMIR5/CD300LB, CX3CR1, CX3CL1, LMIR6/CD300LE,CXCR4, LRP-1, CXCR6, M-CSF R, DEP-1/CD148, MD-1, DNAM-1, MD-2,EMMPRIN/CD147, MMR, Endoglin/CD105, NCAM-L1, Fc gamma R1/CD64, PSGL-1,Fc gamma RIII/CD16, RP105, G-CSF R, L-Selectin, GM-CSF R alpha,Siglec-3/CD33, HVEM/TNFRSF14, SLAM, ICAM-1/CD54, TCCR/WSX-1,ICAM-2/CD102, TREM-1, IL-6 R, TREM-2, CXCR1/IL-8 RA, TREM-3 andTREML1/TLT-1.

In yet other embodiments of the method, a Dendritic cell target isselected from the group consisting of CD36/SR-B3, LOX-1/SR-E1, CD68,MARCO, CD163, SR-AI/MSR, CD5L, SREC-1, CL-P1/COLEC12, SREC-II,LIMPII/SR-B2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, 4-1BBLigand/TNFSF9, IL-12/IL-23 p40, 4-Amino-1,8-naphthalimide, ILT2/CD85j,CCL21/6Ckine, ILT3/CD85k, 8-oxo-dG, ILT4/CD85d, 8D6A, ILT5/CD85a, A2B5,Integrin alpha 4/CD49d, Aag, Integrin beta 2/CD18, AMICA, Langerin,B7-2/CD86, Leukotriene B4 R1, B7-H3, LMIR1/CD300A, BLAME/SLAMF8,LMIR2/CD300c, C1q R1/CD93, LMIR3/CD300LF, CCR6, LMIR5/CD300LB, CCR7,LMIR6/CD300LE, CD40/TNFRSF5, MAG/Siglec-4a, CD43, MCAM, CD45, MD-1,CD68, MD-2, CD83, MDL-1/CLEC5A, CD84/SLAMF5, MMR, CD97, NCAM-L1,CD2F-10/SLAMF9, Osteoactivin/GPNMB, Chem 23, PD-L2, CLEC-1, RP105,CLEC-2, Siglec-2/CD22, CRACC/SLAMF7, Siglec-3/CD33, DC-SIGN, Siglec-5,DC-SIGNR/CD299, Siglec-6, DCAR, Siglec-7, DC1R/CLEC4A, Siglec-9,DEC-205, Siglec-10, Dectin-1/CLEC7A, Siglec-F, Dectin-2/CLEC6A,SIGNR1/CD209, DEP-1/CD148, SIGNR4, DLEC, SLAM, EMMPRIN/CD147,TCCR/WSX-1, Fc gamma R1/CD64, TLR₃, Fc gamma RIIB/CD32b, TREM-1, Fcgamma RIIC/CD32c, TREM-2, Fc gamma RIIA/CD32a, TREM-3, Fc gammaRIII/CD16, TREML1/TLT-1, ICAM-2/CD102 and Vanilloid R1.

In still other embodiments of the method, the angiogenesis target isselected from the group consisting of Angiopoietin-1, Angiopoietin-like2, Angiopoietin-2, Angiopoietin-like 3, Angiopoietin-3,Angiopoietin-like 7/CDT6, Angiopoietin-4, Tie-1, Angiopoietin-like 1,Tie-2, Angiogenin, iNOS, Coagulation Factor III/Tissue Factor, nNOS,CTGF/CCN2, NOV/CCN3, DANCE, OSM, EDG-1, Plfr, EG-VEGF/PK1, Proliferin,Endostatin, ROBO4, Erythropoietin, Thrombospondin-1, Kininostatin,Thrombospondin-2, MFG-E8, Thrombospondin-4, Nitric Oxide, VG5Q, eNOS,EphA1, EphA5, EphA2, EphA6, EphA3, EphA7, EphA4, EphA8, EphB1, EphB4,EphB2, EphB6, EphB3, Ephrin-A1, Ephrin-A4, Ephrin-A2, Ephrin-A5,Ephrin-A3, Ephrin-B1, Ephrin-B3, Ephrin-B2, FGF acidic, FGF-12, FGFbasic, FGF-13, FGF-3, FGF-16, FGF-4, FGF-17, FGF-5, FGF-19, FGF-6,FGF-20, FGF-8, FGF-21, FGF-9, FGF-23, FGF-10, KGF/FGF-7, FGF-11, FGF R1,FGF R4, FGF R2, FGF R5, FGF R3, Neuropilin-1, Neuropilin-2, Semaphorin3A, Semaphorin 6B, Semaphorin 3C, Semaphorin 6C, Semaphorin 3E,Semaphorin 6D, Semaphorin 6A, Semaphorin 7A, MMP, MMP-11, MMP-1, MMP-12,MMP-2, MMP-13, MMP-3, MMP-14, MMP-7, MMP-15, MMP-8, MMP-16/MT3-MMP,MMP-9, MMP-24/MT5-MMP, MMP-10, MMP-25/MT6-MMP, TIMP-1, TIMP-3, TIMP-2,TIMP-4, ACE, IL-13 R alpha 1, IL-13, C1q R1/CD93, Integrin alpha4/CD49d, VE-Cadherin, Integrin beta 2/CD18, CD31/PECAM-1, KLF4,CD36/SR-B3, LYVE-1, CD151, MCAM, CL-P1/COLEC12, Nectin-2/CD112,Coagulation Factor III/Tissue Factor, E-Selectin, D6, P-Selectin,DC-SIGNR/CD299, SLAM, EMMPRIN/CD147, Tie-2, Endoglin/CD105, TNFRI/TNFRSF1A, EPCR, TNF RII/TNFRSF1B, Erythropoietin R, TRAILR1/TNFRSF10A, ESAM, TRAIL R2/TNFRSF10B, FABP5, VCAM-1, ICAM-1/CD54, VEGFR2/Flk-1, ICAM-2/CD102, VEGF R3/Flt-4, IL-1 RI and VG5Q.

Other embodiments of the method provide multivalent binding proteinswherein at least one of binding domain 1 and binding domain 2specifically binds a target selected from the group consisting ofProstate-specific Membrane Antigen (Folate Hydrolase 1), EpidermalGrowth Factor Receptor (EGFR), Receptor for Advanced Glycation Endproducts (RAGE, also known as Advanced Glycosylation End productReceptor or AGER), IL-17 A, IL-17 F, P19 (IL23A and IL12B), Dickkopf-1(Dkk1), NOTCH1, NG2 (Chondroitin Sulfate ProteoGlycan 4 or CSPG4), IgE(IgHE or IgH2), IL-22R (IL22RA1), IL-21, Amyloid β oligomers (Aboligomers), Amyloid β Precursor Protein (APP), NOGO Receptor (RTN4R),Low Density LipoproteinReceptor-Related Protein 5 (LRP5), IL-4,Myostatin (GDF8), Very Late Antigen 4, an alpha 4, beta 1 integrin (VLA4or ITGA4), an alpha 4, beta 7 integrin found on leukocytes, and IGF-1R.For example, a VLA4 target may be recognized by a multivalent bindingprotein in which at least one of binding domain 1 and binding domain 2is a binding domain derived from Natalizumab (Antegren).

In some embodiments, the cancer cell is a transformed, or cancerous,hematopoietic cell. In certain of these embodiments, at least one of thefirst binding domain and the second binding domain recognizes a targetselected from the group consisting of a B-cell target, amonocyte/macrophage target, a dendritic cell target, an NK-cell targetand a T-cell target, each as herein defined. Further, at least one ofthe first binding domain and the second binding domain can recognize amyeloid targets, including but not limited to, CD5, CD10, CD11b, CD11c,CD13, CD14, CD15, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD29,CD30, CD31, CD33, CD34, CD35, CD38, CD43, CD45, CD64, CD66, CD68, CD70,CD80, CD86, CD87, CD88, CD89, CD98, CD100, CD103, CD111, CD112, CD114,CD115, CD116, CD117, CD118, CD119, CD120a, CD120b, CDw123, CDw131,CD141, CD162, CD163, CD177, CD312, IRTA1, IRTA2, IRTA3, IRTA4, IRTA5,B-B2, B-B8 and B-cell antigen receptor.

Other embodiments of the invention are drawn to the multivalent bindingprotein, as described herein, comprising a sequence selected from thegroup consisting of SEQ ID NOS:2, 4, 6, 103, 105, 107, 109, 332, 333,334, and 345. Other embodiments are directed to the multivalent bindingprotein comprising a sequence selected from the group consisting of SEQID NOS:355, 356, 357, 358, 359, 360, 361, 362, 363, 364 and 365.

In other embodiments, the multivalent and multispecific binding proteinwith effector function has a first binding domain and a second bindingdomain that recognize a target pair selected from the group consistingof EPHB4-KDR and TIE-TEK. In such embodiments, the protein has a firstbinding domain recognizing EPHB4 and a second binding domain recognizingKDR or a first binding domain recognizing KDR and a second bindingdomain recognizing EPHB4. Analogously, the protein may have a firstbinding domain recognizing TIE and a second binding domain recognizingTEK, or a first binding domain recognizing TEK and a second bindingdomain recognizing TIE.

In a related aspect, the invention provides a multivalent bindingprotein with effector function, wherein the constant sub-regionrecognizes an effector cell F_(C) receptor (e.g., F_(C)γRI, F_(C)γRII,F_(C)γRIII, F_(C)αR, and F_(C)εRI. In particular embodiments, theconstant sub-region recognizes an effector cell surface protein selectedfrom the group consisting of CD2, CD3, CD16, CD28, CD32, CD40, CD56,CD64, CD89, F_(Cε)RI, KTR, thrombospondin R, NKG2D, 2B4/NAIL and 41BB.The constant sub-region may comprise a C_(H2) domain and a C_(H3) domainderived from the same, or different, immunoglobulins, antibody isotypes,or allelic variants. In some embodiments, the C_(H3) domain is truncatedand comprises a C-terminal sequence selected from the group consistingof SEQ ID NOS: 366, 367, 368, 369, 370 and 371. Preferably, the C_(H2)domain and the scorpion linker are derived from the same class, or fromthe same sub-class, of immunoglobulin, when the linker is a hinge-likepeptide derived from an immunoglobulin.

Some proteins according to the invention are also contemplated asfurther comprising a scorpion linker of at least about 5 amino acidsattached to the constant sub-region and attached to the second bindingdomain, thereby localizing the scorpion linker between the constantsub-region and the second binding domain. Typically, the scorpion linkerpeptide length is between 5-45 amino acids. Scorpion linkers includehinge-like peptides derived from immunoglobulin hinge regions, such asIgG1, IgG2, IgG3, IgG4, IgA, and IgE hinge regions. Preferably, ahinge-like scorpion linker will retain at least one cysteine capable offorming an interchain disulfide bond under physiological conditions.Scorpion linkers derived from IgG1 may have 1 cysteine or two cysteines,and will preferably retain the cysteine corresponding to an N-terminalhinge cysteine of IgG1. In some embodiments, the scorpion linker isextended relative to a cognate immunoglobulin hinge region and, inexemplary embodiments, comprises a sequence selected from the groupconsisting of SEQ ID NOS:351, 352, 353 and 354. Non-hinge-like peptidesare also contemplated as scorpion linkers, provided that such peptidesprovide sufficient spacing and flexibility to provide a single-chainprotein capable of forming two binding domains, one located towards eachprotein terminus (N and C) relative to a more centrally located constantsub-region domain. Exemplary non-hinge-like scorpion linkers includepeptides from the stalk region of type II C-lectins, such as the stalkregions of CD69, CD72, CD94, NKG2A and NKG2D. In some embodiments, thescorpion linker comprises a sequence selected from the group consistingof SEQ ID NOS:373, 374, 375, 376 and 377.

The proteins may also comprise a linker of at least about 5 amino acidsattached to the constant sub-region and attached to the first bindingdomain, thereby localizing the linker between the constant sub-regionand the first binding domain. In some embodiments, linkers are foundbetween the constant sub-region and each of the two binding domains, andthose linkers may be of the same or different sequence, and of the sameor different lengths.

The constant sub-region of the proteins according to the inventionprovides at least one effector function. Any effector function known inthe art to be associated with an immunoglobulin (e.g., an antibody) iscontemplated, such as an effector function selected from the groupconsisting of antibody-dependent cell-mediated cytotoxicity (ADCC),complement-dependent cytotoxicity (CDC), relatively extended in vivohalf-life (relative to the same molecule lacking a constant sub-region),FcR binding, protein A binding, and the like. In some embodiments, theextended half-lives of proteins of the invention are at least 28 hoursin a human. Of course, proteins intended for administration to non-humansubjects will exhibit relatively extended half-lives in those non-humansubjects, and not necessarily in humans.

In general, the proteins (including polypeptides and peptides) of theinvention exhibit a binding affinity of less than 10⁻⁹ M, or at least10⁻⁶ M, for at least one of the first binding domain and the secondbinding domain.

Another aspect of the invention is drawn to a pharmaceutical compositioncomprising a protein as described herein and a pharmaceuticallyacceptable adjuvant, carrier or excipient. Any adjuvant, carrier, orexcipient known in the art is useful in the pharmaceutical compositionsof the invention.

Yet another aspect of the invention provides a method of producing aprotein as described above comprising introducing a nucleic acidencoding the protein into a host cell and incubating the host cell underconditions suitable for expression of the protein, thereby expressingthe protein, preferably at a level of at least 1 mg/liter. In someembodiments, the method further comprises isolating the protein byseparating it from at least one protein with which it is associated uponintracellular expression. Suitable host cells for expressing the nucleicacids to produce the proteins of the invention include, but are notlimited to, a host cell selected from the group consisting of a VEROcell, a HeLa cell, a CHO cell, a COS cell, a W138 cell, a BHK cell, aHepG2 cell, a 3T3 cell, a RIN cell, an MDCK cell, an A549 cell, a PC12cell, a K562 cell, a HEK293 cell, an N cell, a Spodoptera frugiperdacell, a Saccharomyces cerevisiae cell, a Pichia pastoris cell, any of avariety of fungal cells and any of a variety of bacterial cells(including, but not limited to, Escherichia coli, Bacillus subtilis,Salmonella typhimurium, and a Streptomycete).

The invention also provides a method of producing a nucleic acidencoding the protein, as described above, comprising covalently linkingthe 3′ end of a polynucleotide encoding a first binding domain derivedfrom an immunoglobulin variable region to the 5′ end of a polynucleotideencoding a constant sub-region, covalently linking the 5′ end of apolynucleotide encoding a scorpion linker to the 3′ end of thepolynucleotide encoding the constant sub-region, and covalently linkingthe 5′ end of a polynucleotide encoding a second binding domain derivedfrom an immunoglobulin variable region to the 3′ end of thepolynucleotide encoding the scorpion linker, thereby generating anucleic acid encoding a multivalent binding protein with effectorfunction. Each of these coding regions may be separated by a codingregion for a linker or hinge-like peptide as part of a single-chainstructure according to the invention. In some embodiments, the methodproduces a polynucleotide encoding a first binding domain that comprisesa sequence selected from the group consisting of SEQ ID NO: 2 (anti-CD20variable region, oriented V_(L)-V_(H)), SEQ ID NO: 4 (anti-CD28 variableregion, oriented V_(L)-V_(H)) and SEQ ID NO: 6 (anti-CD28 variableregion, oriented V_(H)-V_(L)) in single-chain form, rather thanrequiring assembly from separately encoded polypeptides as must occurfor heteromultimeric proteins, including natural antibodies. Exemplarypolynucleotide sequences encoding first binding domains arepolynucleotides comprising any of SEQ ID NOS: 1, 3 or 5.

This aspect of the invention also provides methods for producingencoding nucleic acids that further comprise a linker polynucleotideinserted between the polynucleotide encoding a first binding domain andthe polynucleotide encoding a constant sub-region, the linkerpolynucleotide encoding a peptide linker of at least 5 amino acids.Additionally, these methods produce nucleic acids that further comprisea linker polynucleotide inserted between the polynucleotide encoding aconstant sub-region and the polynucleotide encoding a second bindingdomain, the linker polynucleotide encoding a peptide linker of at least5 amino acids. Preferably, the encoded peptide linkers are between 5 and45 amino acids.

The identity of the linker regions present either between BD1 and EFD orEFD and BD2 may be derived from other sequences identified fromhomologous -Ig superfamily members. In developing novel linkers derivedfrom existing sequences present in homologous members of the -Igsuperfamily, it may be preferable to avoid sequence stretches similar tothose located between the end of a C-like domain and the transmembranedomain, since such sequences are often substrates for protease cleavageof surface receptors from the cell to create soluble forms. Sequencecomparisons between different members of the -Ig superfamily andsubfamilies can be compared for similarities between molecules in thelinker sequences that join multiple V-like domains or between the V andC like domains. From this analysis, conserved, naturally occurringsequence patterns may emerge; these sequences when used as the linkersbetween subdomains of the multivalent fusion proteins should be moreprotease resistant, might facilitate proper folding between Ig loopregions, and would not be immunogenic since they occur in theextracellular domains of endogenous cell surface molecules.

The nucleic acids themselves comprise another aspect of the invention.Contemplated are nucleic acids encoding any of the proteins of theinvention described herein. As such, the nucleic acids of the inventioncomprise, in 5′ to 3′ order, a coding region for a first binding domain,a constant sub-region sequence, and a coding region for a second bindingdomain. Also contemplated are nucleic acids that encode protein variantswherein the two binding domains and the constant sub-region sequencesare collectively at least 80%, and preferably at least 85%, 90%, 95%, or99% identical in amino acid sequence to the combined sequences of aknown immunoglobulin variable region sequence and a known constantsub-region sequence. Alternatively, the protein variants of theinvention are encoded by nucleic acids that hybridize to a nucleic acidencoding a non-variant protein of the invention under stringenthybridization conditions of 0.015 M sodium chloride, 0.0015 M sodiumcitrate at 65-68° C. or 0.015 M sodium chloride, 0.0015 M sodiumcitrate, and 50% formamide at 42° C. Variant nucleic acids of theinvention exhibit the capacity to hybridize under the conditions definedimmediately above, or exhibit 90%, 95%, 99%, or 99.9% sequence identityto a nucleic acid encoding a non-variant protein according to theinvention.

In related aspects, the invention provides a vector comprising a nucleicacid as described above, as well as host cells comprising a vector or anucleic acid as described herein. Any vector known in the art may beused (e.g., plasmids, phagemids, phasmids, cosmids, viruses, artificialchromosomes, shuttle vectors and the like) and those of skill in the artwill recognize which vectors are particularly suited for a givenpurpose. For example, in methods of producing a protein according to theinvention, an expression vector operable in the host cell of choice isselected. In like manner, any host cell capable of being geneticallytransformed with a nucleic acid or vector of the invention iscontemplated. Preferred host cells are higher eukaryotic host cells,although lower eukaryotic (e.g., yeast) and prokaryotic (bacterial) hostcells are contemplated.

Another aspect of the invention is drawn to a method of inducing damageto a target cell comprising contacting a target cell with atherapeutically effective amount of a protein as described herein. Insome embodiments, the target cell is contacted in vivo by administrationof the protein, or an encoding nucleic acid, to an organism in need.Contemplated within this aspect of the invention are methods wherein themultivalent single-chain binding protein induces an additive amount ofdamage to the target cell, which is that amount of damage expected fromthe sum of the damage attributable to separate antibodies comprising oneor the other of the binding domains. Also contemplated are methodswherein the multivalent single-chain binding protein induces asynergistic amount of damage to the target cell compared to the sum ofthe damage induced by a first antibody comprising the first bindingdomain but not the second binding domain and a second antibodycomprising the second binding domain but not the first binding domain.In some embodiments, the multivalent single-chain binding protein ismultispecific and comprises a binding domain pair specificallyrecognizing a pair of antigens selected from the group consisting ofCD19/CD20, CD20/CD21, CD20/CD22, CD20/CD40, CD20/CD79a, CD20/CD79b,CD20/CD81, CD21/CD79b, CD37/CD79b, CD79b/CD81, CD19/CL II (i.e., MHCclass II), CD20/CL II, CD30/CL II, CD37/CL II, CD72/CL II, and CD79b/CLII.

This aspect of the invention also comprehends methods wherein themultispecific, multivalent single-chain binding protein induces aninhibited amount of damage to the target cell compared to the sum of thedamage induced by a first antibody comprising the first binding domainbut not the second binding domain and a second antibody comprising thesecond binding domain but not the first binding domain. Exemplaryembodiments include methods wherein the multi-specific, multivalentsingle-chain binding protein comprises a binding domain pairspecifically recognizing a pair of antigens selected from the groupconsisting of CD20/CL II, CD21/CD79b, CD22/CD79b, CD40/CD79b,CD70/CD79b, CD72/CD79b, CD79a/CD79b, CD79b/CD80, CD79b/CD86, CD21/CL II,CD22/CL II, CD23/CL II, CD40/CL II, CD70/CL II, CD80/CL II, CD86/CL II,CD19/CD22, CD20/CD22, CD21/CD22, CD22/CD23, CD22/CD30, CD22/CD37,CD22/CD40, CD22/CD70, CD22/CD72, CD22/79a, CD22/79b, CD22/CD80,CD22/CD86 and CD22/CL II.

In a related aspect, the invention provides a method of treating a cellproliferation disorder, e.g., cancer, comprising administering atherapeutically effective amount of a protein (as described herein), oran encoding nucleic acid, to an organism in need. Those of skill in theart, including medical and veterinary professionals, are proficient atidentifying organisms in need of treatment. Disorders contemplated bythe invention as amenable to treatment include a disorder selected fromthe group consisting of a cancer, an autoimmune disorder, Rous SarcomaVirus infection and inflammation. In some embodiments, the protein isadministered by in vivo expression of a nucleic acid encoding theprotein as described herein. The invention also comprehendsadministering the protein by a route selected from the group consistingof intravenous injection, intraarterial injection, intramuscularinjection, subcutaneous injection, intraperitoneal injection and directtissue injection.

Another aspect of the invention is directed to a method of amelioratinga symptom associated with a cell proliferation disorder comprisingadministering a therapeutically effective amount of a protein, asdescribed herein, to an organism in need. Those of skill in the art arealso proficient at identifying those disorders, or diseases orconditions, exhibiting symptoms amenable to amelioration. In someembodiments, the symptom is selected from the group consisting of pain,heat, swelling and joint stiffness.

Yet another aspect of the invention is drawn to a method of treating aninfection associated with an infectious agent comprising administering atherapeutically effective amount of a protein according to the inventionto a patient in need, wherein the protein comprises a binding domainthat specifically binds a target molecule of the infectious agent.Infectious agents amenable to treatment according to this aspect of theinvention include prokaryotic and eukaryotic cells, viruses (includingbacteriophage), foreign objects, and infectious organisms such asparasites (e.g., mammalian parasites).

A related aspect of the invention is directed to a method ofameliorating a symptom of an infection associated with an infectiousagent comprising administering an effective amount of a proteinaccording to the invention to a patient in need, wherein the proteincomprises a binding domain that specifically binds a target molecule ofthe infectious agent. Those of skill in the medical and veterinary artswill be able to determine an effective amount of a protein on acase-by-case basis, using routine experimentation.

Yet another related aspect of the invention is a method of reducing therisk of infection attributable to an infectious agent comprisingadministering a prophylactically effective amount of a protein accordingto the invention to a patient at risk of developing the infection,wherein the protein comprises a binding domain that specifically binds atarget molecule of the infectious agent. Those of skill in the relevantarts will be able to determine a prophylactically effective amount of aprotein on a case-by-case basis, using routine experimentation.

Another aspect of the invention is drawn to the above-describedmultivalent single-chain binding protein wherein at least one of thefirst binding domain and the second binding domain specifically binds anantigen selected from the group consisting of CD19, CD20, CD21, CD22,CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86, anda major histocompatibility complex class II peptide.

In certain embodiments, one of the first binding domain and the secondbinding domain specifically binds CD20, and in some of theseembodiments, the other binding domain specifically binds an antigenselected from the group consisting of CD19, CD20, CD21, CD22, CD23,CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86, and amajor histocompatibility complex class II peptide. For example, in oneembodiment, the first binding domain is capable of specifically bindingCD20 while the second binding domain is capable of specifically binding,e.g., CD19. In another embodiment, the first binding domain binds CD19while the second binding domain binds CD20. An embodiment in which bothbinding domains bind CD20 is also contemplated.

In certain other embodiments according to this aspect of the invention,one of the first binding domain and the second binding domainspecifically binds CD79b, and in some of these embodiments, the otherbinding domain specifically binds an antigen selected from the groupconsisting of CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70,CD72, CD79a, CD79b, CD80, CD81, CD86, and a major histocompatibilitycomplex class II peptide. Exemplary embodiments include distinctmulti-specific, multivalent single-chain binding proteins in which afirst binding domain:second binding domain specifically binds CD79b:CD19or CD19:CD79b. A multivalent binding protein having first and secondbinding domains recognizing CD79b is also comprehended.

In still other certain embodiments, one of the first binding domain andthe second binding domain specifically binds a major histocompatibilitycomplex class II peptide, and in some of these embodiments, the otherbinding domain specifically binds an antigen selected from the groupconsisting of CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70,CD72, CD79a, CD79b, CD80, CD81, CD86, and a major histocompatibilitycomplex class II peptide. For example, in one embodiment, the firstbinding domain is capable of specifically binding a majorhistocompatibility complex class II peptide while the second bindingdomain is capable of specifically binding, e.g., CD19. In anotherembodiment, the first binding domain binds CD19 while the second bindingdomain binds a major histocompatibility complex class II peptide. Anembodiment in which both binding domains bind a major histocompatibilitycomplex class II peptide is also contemplated.

In yet other embodiments according to this aspect of the invention, oneof the first binding domain and the second binding domain specificallybinds CD22, and in some of these embodiments, the other binding domainspecifically binds an antigen selected from the group consisting ofCD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a,CD79b, CD80, CD81, CD86, and a major histocompatibility complex class IIpeptide. Exemplary embodiments include distinct multi-specific,multivalent single-chain binding proteins in which a first bindingdomain:second binding domain specifically binds CD22:CD19 or CD19:CD22.A multivalent binding protein having first and second binding domainsrecognizing CD22 is also comprehended.

A related aspect of the invention is directed to the above-describedmultivalent single-chain binding protein wherein the protein has asynergistic effect on a target cell behavior relative to the sum of theeffects of each of the binding domains. In some embodiments, the proteincomprises a binding domain pair specifically recognizing a pair ofantigens selected from the group consisting of CD20-CD19, CD20-CD21,CD20-CD22, CD20-CD40, CD20-CD79a, CD20-CD79b and CD20-CD81.

The invention further comprehends a multivalent single-chain bindingprotein as described above wherein the protein has an additive effect ona target cell behavior relative to the sum of the effects of each of thebinding domains. Embodiments according to this aspect of the inventioninclude multi-specific proteins comprising a binding domain pairspecifically recognizing a pair of antigens selected from the groupconsisting of CD20-CD23, CD20-CD30, CD20-CD37, CD20-CD70, CD20-CD80,CD20-CD86, CD79b-CD37, CD79b-CD81, major histocompatibility complexclass II peptide-CD30, and major histocompatibility complex class IIpeptide-CD72.

Yet another related aspect of the invention is a multivalentsingle-chain binding protein as described above wherein the protein hasan inhibitory effect on a target cell behavior relative to the sum ofthe effects of each of the binding domains. In some embodiments, theprotein is multispecific and comprises a binding domain pairspecifically recognizing a pair of antigens selected from the groupconsisting of CD20-major histocompatibility complex class II peptide,CD79b-CD19, CD79b-CD20, CD79b-CD21, CD79b-CD22, CD79b-CD23, CD79b-CD30,CD79b-CD40, CD79b-CD70, CD79b-CD72, CD79b-CD79a, CD79b-CD80, CD79b-CD86,CD79b-major histocompatibility complex class II peptide, majorhistocompatibility complex class II peptide-CD19, majorhistocompatibility complex class II peptide-CD20, majorhistocompatibility complex class II peptide-CD21, majorhistocompatibility complex class II peptide-CD22, majorhistocompatibility complex class II peptide-CD23, majorhistocompatibility complex class II peptide-CD37, majorhistocompatibility complex class II peptide-CD40, majorhistocompatibility complex class II peptide-CD70, majorhistocompatibility complex class II peptide-CD79a, majorhistocompatibility complex class II peptide-CD79b, majorhistocompatibility complex class H peptide-CD80, majorhistocompatibility complex class II peptide-CD81, majorhistocompatibility complex class II peptide-CD86, CD22-CD19, CD22-CD40,CD22-CD79b, CD22-CD86 and CD22-major histocompatibility complex class IIpeptide.

Another aspect of the invention is a method of identifying at least oneof the binding domains of the multivalent binding molecule, such as amultispecific binding molecule, described above comprising: (a)contacting an anti-isotypic antibody with an antibody specificallyrecognizing a first antigen and an antibody specifically recognizing asecond antigen; (b) further contacting a target comprising at least oneof said antigens with the composition of step (a); and (c) measuring anactivity of the target, wherein the activity is used to identify atleast one of the binding domains of the multivalent binding molecule. Insome embodiments, the target is a diseased cell, such as a cancer cell(e.g., a cancerous B-cell) or an auto-antibody-producing B-cell.

In each of the foregoing methods of the invention, it is contemplatedthat the method may further comprise a plurality of multivalentsingle-chain binding proteins. In some embodiments, a binding domain ofa first multivalent single-chain binding protein and a binding domain ofa second multivalent single-chain binding protein induce a synergistic,additive, or inhibitory effect on a target cell, such as a synergistic,additive, or inhibitory amount of damage to the target cell. Thesynergistic, additive or inhibitory effects of a plurality ofmultivalent single-chain binding proteins is determined by comparing theeffect of such a plurality of proteins to the combined effect of anantibody comprising one of the binding domains and an antibodycomprising the other binding domain.

A related aspect of the invention is directed to a compositioncomprising a plurality of multivalent single-chain binding proteins asdescribed above. In some embodiments, the composition comprises aplurality of multivalent single-chain binding proteins wherein a bindingdomain of a first multivalent single-chain binding protein and a bindingdomain of a second multivalent single-chain binding protein are capableof inducing a synergistic, additive, or inhibitory effect on a targetcell, such as a synergistic, additive or inhibitory amount of damage tothe target cell.

The invention further extends to a pharmaceutical composition comprisingthe composition described above and a pharmaceutically acceptablecarrier, diluent or excipient. In addition, the invention comprehends akit comprising the composition as described herein and a set ofinstructions for administering said composition to exert an effect on atarget cell, such as to damage the target cell.

Finally, the invention also comprehends a kit comprising the protein asdescribed herein and a set of instructions for administering the proteinto treat a cell proliferation disorder or to ameliorate a symptom of thecell proliferation disorder.

Other features and advantages of the present invention will be betterunderstood by reference to the following detailed description, includingthe examples.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows a schematic representation of the multivalent single-chainmolecules envisioned by the invention. Individual subdomains of thefusion protein expression cassette are indicated by separateshapes/blocks on the figure. BD1 refers to binding domain 1, linker 1refers to any potential linker or hinge like peptide between BD1 and the“effector function domain”, indicated as EFD. This subdomain is usuallyan engineered form of the Fc domain of human IgG1, but may include othersubdomains with one or more effector functions as defined herein. Linker2 refers to the linker sequence, if any, present between the carboxyterminus of the EFD and the binding domain 2, BD2.

FIG. 2 shows a Western blot of non-reduced proteins expressed in COScells. Protein was secreted into the culture medium, and culturesupernatants isolated after 48-72 hours from transiently transfectedcells by centrifugation. Thirty microliters, 30 μl of crude supernatantwere loaded into each well of the gel. Lane identifications: 1—molecularweight markers, with numerals indicating kilodaltons;2-2H7-IgG-STD1-2E12 LH; 3-2H7-IgG-STD1-2E12 HL, 4-2H7-IgG-STD2-2E12 LH;5-2H7-IgG-STD2-2E12 HL; 6-2E12 LH SMIP; 7-2E12 HL SMIP; 8-2H7 SMIP.“2H7” refers to a single-chain construct, where BD 1 encodes the CD20specific binding domain (2H7) in the VLVH orientation; “2E12” refers toa binding domain specific for CD28; -IgG-refers to a single-chainconstruct, with a hinge encoding a sequence where all C are mutated to S(sss), and the CH2 and CH3 domains of IgG1 contain mutations whicheliminate both ADCC and CDC effector functions (P238S and P331 S), “STD1 refers to a 20-amino-acid linker (identified in FIG. 7 as “STD1=20aa”)inserted adjacent to the BD2 in the VL-VH orientation, or 2E12(V_(L)-V_(H)). “STD1-HL” refers to a similar construct as justdescribed, but with the BD2 V regions in the VH-VL orientation asfollows: 2H7-sssIgG (P238/331S)-20-amino-acid linker-2E12 (V_(H)-V_(L)).“STD2-LH” refers to 2H7-sssIgG (P238/331S)-38-amino-acid linker-2E12(V_(L)-V_(H)); “STD2-LH” refers to 2H7-sssIgG (P238/331SS)-38-amino-acidlinker-2E12 (V_(H)-V_(L)); “SMIP” refers to small modularimmunopharmaceutical; and “H” generally refers to V_(H), while “L”generally refers to V_(L). Unless otherwise indicated, all proteinorientations are N-terminal to C-terminal orientations.

FIG. 3 shows two columnar graphs illustrating the binding properties ofthe 2H7-sssIgG (P238S/P331S)-STD1-2e12 LH and HL derivatives expressedfrom COS cells. These experiments were performed with crude culturesupernatants rather than purified proteins. Serial dilutions fromundiluted to 16× of the culture supernatants were incubated with eitherCD20 expressing cells (WIL-2S) or CD28 expressing cells (CD28 CHO).Binding activity in the supernatants was compared to control samplestesting binding of the relevant single specificity SMIP, such asTRU-015, or 2e12 VLVH, or 2e12VHVL SMIPs. Binding in each sample wasdetected using fluorescein isothyocyanate (FITC) conjugated goatanti-human IgG at a dilution of 1:100.

FIG. 4 is a histogram showing the binding pattern of protein A purifiedversions of the proteins tested in FIG. 3 to WIL2-S cells. “TRU015” is aSMIP specific for CD20. Two multispecific binding proteins with effectorfunction were also analyzed: “2H7-2E12 LH” has binding domain 2,specific for CD28, in V_(L)-V_(H) orientation; “2H7-2E12 HL” has bindingdomain 2, specific for CD28, in V_(H)-V_(L) orientation. Each of theproteins was tested for binding at 5 μg/ml, and binding detected withFITC goat anti-human IgG at 1:100. See the description for FIG. 2 abovefor more complete descriptions of the molecules tested.

FIG. 5 shows two histograms illustrating the binding by protein Apurified multispecific binding proteins with effector function to CHOcells expressing CD28. “2H7-2E12 LH” has binding domain 2, specific forCD28, in V_(L)-V_(H) orientation; “2H7-2E12 HL” has binding domain 2,specific for CD28, in V_(H)-V_(L) orientation. Each of the proteins wastested for binding at 5 μg/ml, and binding was detected with FITC goatanti-human IgG at 1:100. See the descriptions in FIG. 2 for a morecomplete description of the molecules tested.

FIG. 6 A) shows a table which identifies the linkers joining theconstant sub-region and binding domain 2. The linkers are identified byname, sequence, sequence identifier, sequence length, and the sequenceof the fusion with binding domain 2. B) shows a table identifying avariety of constructs identifying elements of exemplified moleculesaccording to the invention. In addition to identifying the multivalentbinding molecules by name, the elements of those molecules are disclosedin terms of binding domain 1 (BD1), the constant sub-region (hinge andeffector domain or EFD), a linker (see FIG. 6A for additionalinformation regarding the linkers), and binding domain 2 (BD2). Thesequences of a number of exemplified multivalent binding proteins areprovided, and are identified in the figure by a sequence identifier.Other multivalent binding proteins have altered elements, or elementorders, with predictable alterations in sequence from the disclosedsequences.

FIG. 7 shows a composite columnar graph illustrating the binding ofpurified proteins at a single, fixed concentration to CD20 expressingWIL-2S cells and to CHO cells expressing CD28. “H1-H6” refers to the2H7-sss-hIgG-Hx-2e12 molecules with the H1-H6 linkers and the 2e12 Vregions in the orientation of V_(H)-V_(L). “L1-L6” refers to the2H7-sss-hIgG-Lx-2e12 molecules with the L1-L6 linkers and the 2e12 Vregions in the orientation of V_(L)-V_(H). All the molecules were testedat a concentration of 0.72 μg/ml, and the binding detected using FITCconjugated goat anti-human IgG at 1:100. The mean fluorescence intensityfor each sample was then plotted as paired bar graphs for the two targetcell types tested versus each of the multivalent constructs beingtested, L1-L6, or H1-H6.

FIG. 8 shows photographs of Coomassie stained non-reducing and reducingSDS-PAGE gels. These gels show the effect of the variant linkersequence/length on the 2H7-sss-hIgG-Hx-2e12 HL protein on the amounts ofthe two predominate protein bands visualized on the gel.

FIG. 9 shows Western Blots of the [2H7-sss-hIgG-H6-2e12] fusion proteinsand the relevant single specificity SMIPs probed with either (a)CD28mIgG or with (b) a Fab reactive with the 2H7 specificity. Theresults show that the presence of the H6 linker results in thegeneration of cleaved forms of the multivalent constructs which aremissing the CD28 binding specificity.

FIG. 10 shows binding curves of the different linker variants for the[TRU015-sss-IgG-Hx-2e12 HL] H1-H6 linker forms. The first panel showsthe binding curves for binding to CD20 expressing WIL-2S cells. Thesecond panel shows the binding curves for binding of the different formsto CD28 CHO cells. These binding curves were generated with serialdilutions of protein A purified fusion protein, and binding detectedusing FITC conjugated goat anti-human IgG at 1:100.

FIG. 11 shows a table summarizing the results of SEC fractionation of2H7-sss-IgG-2e12 HL multispecific fusion proteins with variant linkersH1-H7. Each row in the table lists a different linker variant of the[2H7-sss-IgG-Hx-2e12-HL] fusion proteins. The retention time of the peakof interest (POI), and the percentage of the fusion protein present inPOI, and the percentage of protein found in other forms is alsotabulated. The cleavage of the molecules is also listed, with the degreeof cleavage indicated in a qualitative way, with (Yes), Yes, and YES, orNo being the four possible choices.

FIG. 12 shows two graphs with binding curves for [2H7-sss-hIgG-Hx-2e12]multispecific fusion proteins with variant linkers H3, H6, and H7linkers to cells expressing CD20 or CD28. Serial dilutions of theprotein A purified fusion proteins from 10 μg/ml down to 0.005 μg/mlwere incubated with either CD20 expressing WIL-2S cells or CD28 CHOcells. Binding was detected using FITC conjugated goat anti-human IgG at1:100. Panel A shows the binding to WIL-2S cells, and panel B shows thebinding to CD28 CHO cells.

FIG. 13 shows the results of an alternative binding assay generated bythe molecules used for FIG. 12. In this case, the fusion proteins werefirst bound to WIL-2S CD20 expressing cells, and binding was thendetected with CD28mIgG (5 μg/ml) and FITC anti-mouse reagent at 1:100.These results demonstrate the simultaneous binding to both CD20 and CD28in the same molecule.

FIG. 14 shows results obtained using another multispecific fusionconstruct variant. In this case, modifications were made in thespecificity for BD2, so that the V regions for the G28-1 antibody wereused to create a CD37 specific binding domain. Shown are two graphswhich illustrate the relative ability of CD20 and/or CD37 antibodies toblock the binding of the [2H7-sss-IgG-Hx-G28-1] multispecific fusionprotein to Ramos or BJAB cells expressing the CD20 and CD37 targets.Each cell type was preincubated with either the CD20 specific antibody(25 μg/ml) or the CD37 specific antibody (10 μg/ml) or both reagents(these are mouse anti-human reagents) prior to incubation with themultispecific fusion protein. Binding of the multispecific fusionprotein was then detected with a FITC goat anti-human IgG reagent at1:100, (preadsorbed to mouse to eliminate cross-reactivity).

FIG. 15 shows the results of an ADCC assay performed with BJAB targetcells, PBMC effector cells, and with the CD20-hIgG-CD37 specific fusionprotein as the test reagent. For a full description of the procedure seethe appropriate example. The graph plots the concentration of fusionprotein versus the % specific killing at each dosage tested for thesingle specificity SMIP reagents, and for the [2H7-sss-hIgG-STD1-G28-1]LH and HL variants. Each data series plots the dose-response effects forone of these single specificity or multispecific single-chain fusionproteins.

FIG. 16 shows a table tabulating the results of a co-culture experimentwhere PBMC were cultured in the presence of TRU 015, G28-1 SMIP, bothmolecules together, or the [2H7-sss-IgG-H7-G28-1HL] variant. The fusionproteins were used at 20 μg/ml, and incubated for 24 hours or 72 hours.Samples were then stained with CD3 antibodies conjugated to FITC, andeither CD19 or CD40 specific antibodies conjugated to PE, then subjectedto flow cytometry. The percentage of cells in each gate was thentabulated.

FIG. 17 shows two columnar graphs of the effects on B cell lineapoptosis after 24 hour incubation with the [2H7-sss-hIgG-H7-G28-1 HL]molecule or control single CD20 and/or CD37 specificity SMIPs alone orin combination. The percentage of annexin V-propidium iodide positivecells is plotted as a function of the type of test reagent used for thecoincubation experiments. Panel A shows the results obtained using Ramoscells, and panel B shows those for Daudi cells. Each single CD20 or CD37directed SMIP is shown at the concentrations indicated; in addition,where combinations of the two reagents were used, the relative amount ofeach reagent is shown in parentheses. For the multispecific CD20-CD37fusion protein, concentrations of 5, 10, and 20 μg/ml were tested.

FIG. 18 shows two graphs of the [2H7-hIgG-G19-4] molecule variants andtheir binding to either CD3 expressing cells (Jurkats) or CD20expressing cells (WIL-2S). The molecules include[2H7-sss-hIgG-STD1-G19-4 HL], LH, and [2H7-csc-hIgG-STD1-G19-4 HL].Protein A purified fusion proteins were titrated from 20 μg/ml down to0.05 μg/ml, and the binding detected using FITC goat anti-human IgG at1:100. MFI (mean fluorescence intensity) is plotted as a function ofprotein concentration.

FIG. 19 shows the results of ADCC assays performed with the[2H7-hIgG-STD1-G19-4 HL] molecule variants with either an SSS hinge or aCSC hinge, BJAB target cells, and either total human PBMC as effectorcells or NK cell depleted PBMC as effector cells. Killing was scored asa function of concentration of the multispecific fusion proteins. Thekilling observed with these molecules was compared to that seen usingG19-4, TRU 015, or a combination of these two reagents. Each data seriesplots a different test reagent, with the percent specific killingplotted as a function of protein concentration.

FIG. 20 shows the percentage of Ramos B-cells that stained positive withAnnexin V (Ann) and/or propidium iodide (PI) after overnight incubationwith each member of a matrix panel of B-cell antibodies (2 μg/ml) in thepresence, or absence, of an anti-CD20 antibody (present at 2 μg/ml whereadded). Goat-anti-mouse secondary antibody was always present at atwo-fold concentration ratio relative to other antibodies (either matrixantibody alone, or matrix antibody and anti-CD20 antibody). Verticallystriped bars—matrix antibody (2 μg/ml) denoted on X-axis and goatanti-mouse antibody (4 μg/ml). Horizontally striped bars—matrix antibody(2 μg/ml) denoted on X-axis, anti-CD20 antibody (2 μg/ml), and goatanti-mouse antibody (4 μg/ml). The “2^(nd) step” condition served as acontrol and involved the addition of goat anti-mouse antibody at 4 μg/ml(vertically striped bar) or 8 μg/ml (horizontally striped bar), withouta matrix antibody or anti-CD20 antibody. “CL II” (MHC class II) in thefigures refers to a monoclonal antibody cross-reactive to HLA DR, DQ andDP, i.e., to MHC Class II antigens.

FIG. 21 shows the percentage of Ramos B-cells that stained positive withAnnexin V (Ann) and/or propidium iodide (PI) after overnight incubationwith each member of a matrix panel of B-cell antibodies (2 μg/ml) in thepresence, or absence, of an anti-CD79b antibody (present at either 0.5or 1.0 μg/ml where added). See the description of FIG. 20 foridentification of “CL II” and “2^(nd) step” samples. Vertically stripedbars—matrix antibody (2 μg/ml) and goat anti-mouse antibody (4 μg/ml);horizontally striped bars—matrix antibody (2 μg/ml), anti-CD79b antibody(1.0 μg/ml) and goat anti-mouse antibody (6 μg/ml); stippled bars—matrixantibody (2 μg/ml), anti-CD79b antibody (0.5 μg/ml) and goat anti-mouseantibody (5 μg/ml).

FIG. 22 shows the percentage of Ramos B-cells that stained positive withAnnexin V (Ann) and/or propidium iodide (PI) after overnight incubationwith each member of a matrix panel of B-cell antibodies (2 μg/ml) in thepresence, or absence, of an anti-CL II antibody (present at either 0.25or 0.5 μg/ml where added). See the description of FIG. 20 foridentification of “CL II” and “2^(nd) step” samples. Vertically stripedbars—matrix antibody (2 μg/ml) and goat anti-mouse antibody (4 μg/ml);horizontally striped bars—matrix antibody (2 μg/ml), anti-CL II antibody(0.5 μg/ml) and goat anti-mouse antibody (5 μg/ml); stippled bars—matrixantibody (2 μg/ml), anti-CL II antibody (0.25 μg/ml) and goat anti-mouseantibody (4.5 μg/ml).

FIG. 23 shows the percentage of DHL-4 B-cells that stained positive withAnnexin V (Ann) and/or propidium iodide (PI) after overnight incubationwith each member of a matrix panel of B-cell antibodies (2 μg/ml) in thepresence, or absence, of an anti-CD22 antibody (present at 2 μg/ml whereadded). See the description of FIG. 20 for identification of “CL II” and“2^(nd) step” samples. Solid bars—matrix antibody (2 μg/ml) and goatanti-mouse antibody (4 μg/ml); slant-striped bars—matrix antibody (2μg/ml), anti-CD22 antibody (2 μg/ml) and goat anti-mouse antibody (8μg/ml).

FIG. 24 provides a graph demonstrating direct growth inhibition oflymphoma cell lines Su-DHL6 (triangles) and DoHH2 (squares) by free CD20SMIP (closed symbols) or monospecific CD20×CD20 scorpion (open symbols).

FIG. 25 is a graph showing direct growth inhibition of lymphoma celllines Su-DHL-6 (triangles) and DoHH2 (squares) by free anti-CD37 SMIP(closed symbols) or monospecific anti-CD37 scorpion (open symbols).

FIG. 26 presents a graph showing direct growth inhibition of lymphomacell lines Su-DHL-6 (triangles) and DoHH2 (squares) by a combination oftwo different monospecific SMIPs (closed symbols) or by a bispecificCD20-CD37 scorpion (open symbols).

FIG. 27 is a graph revealing direct growth inhibition of lymphoma celllines Su-DHL-6 (triangles) and WSU-NHL (squares) by free CD20 SMIP andCD37 SMIP combination (closed symbols) or bispecific CD20×CD37 scorpion(open symbols).

FIG. 28 provides histograms showing the cell-cycle effects of scorpions.Samples of DoHH2 lymphoma cells were separately left untreated, treatedwith SMIP 016 or treated with the monospecific CD37×CD37 scorpion. Openbars: sub-G₁ phase of the cell cycle; black bars: G₀/G₁ phase; shaded: Sphase; and striped: G₂/M phase.

FIG. 29 presents graphs of data establishing that treatment of lymphomacells with scorpions resulted in increased signaling capacity comparedto free SMIPs, as measured by calcium ion flux.

FIG. 30 provides graphs demonstrating scorpion-dependent cellularcytotoxicity

FIG. 31 shows graphs of data indicating that scorpions mediateComplement Dependent Cytotoxicity.

FIG. 32 provides data in graphical form showing comparative ELISAbinding of a SMIP and a scorpion to low- (B) and high-affinity (A)isoforms of FcγRIII (CD16).

FIG. 33 presents graphs establishing the binding of a SMIP and ascorpion to low (A)- and high (B)-affinity allelotypes of FcγRIII (CD16)in the presence of target cells.

FIG. 34 is a histogram showing the expression level of a CD20×CD20scorpion in two experiments (flask 1 and flask 2) under six differentculturing conditions. Solid black bars: flask 1; striped bars: flask 2.

FIG. 35 provides a histogram showing the production yield of a CD20×CD37scorpion.

FIG. 36 presents SDS-PAGE gels (under reducing and non-reducingconditions) of a SMIP and a scorpion.

FIG. 37 provides a graph showing that scorpions retain the capacity tobind to target cells. Filled squares: CD20 SMIP; filled triangles: CD37SMIP; filled circles: humanized CD20 (2Lm20-4) SMIP; open diamond:CD37×CD37 monospecific scorpion; open squares: CD20×CD37 bi-specificscorpion; and open triangles: humanized CD20 (2Lm20-4)×humanized CD20(2Lm20-4) scorpion.

FIG. 38 contains graphs showing the results of competitive bindingassays establishing that both N- and C-terminal scorpion binding domainsparticipate in target cell binding.

FIG. 39 presents data in the form of graphs showing that scorpions havelower off-rates than SMIPs.

FIG. 40 shows a graph establishing that scorpions are stable in serum invivo, characterized by a reproducible, sustained circulating half-lifefor the scorpion.

FIG. 41 provides a dose-response graph for a CD20×CD37 bispecificscorpion, demonstrating the in vivo efficacy of scorpion administration.

FIG. 42 shows target B-cell binding by a monospecific CD20×CD20 scorpion(S0129) and glycovariants.

FIG. 43 provides graphs illustrating CD20×CD20 scorpions (parent andglycovariants) inducing ADCC-mediated killing of BJAB B-cells.

FIG. 44 shows a gel revealing the effects on scorpion stability arisingfrom changes in the scorpion linker, including changing the sequence ofthat linker and extending the linker by adding an H7 sequence to thelinker, indicated by a “+” in the H7 line under the gel.

FIG. 45 shows the binding to WIL2S cells of a CD20×CD20 scorpion (S0129)and scorpion linker variants thereof.

FIG. 46 shows the direct cell killing of a variety of B-cells by aCD20×CD20 scorpion and by a CD20 SMIP.

FIG. 47 reveals the direct cell killing of additional B-cell lines by amonospecific CD20×CD20 scorpion.

FIG. 48 shows the direct cell killing capacities of each of twomonospecific scorpions, i.e., CD20×CD20 and CD37×CD37, and a bispecificCD20×CD37 scorpion, the latter exhibiting a different form of killcurve.

FIG. 49 graphically depicts the response of Su-DHL-6 B-cells to each ofa CD20×CD20 (S0129), a CD37×CD37, and a CD20×CD37 scorpion.

FIG. 50 shows the capacity of a bispecific CD19×CD37 scorpion andRituxan® to directly kill Su-DHL-6 B-cells.

FIG. 51 provides histograms showing the direct killing of DHL-4 B-cellsby a variety of CD20-binding scorpions and SMIPs, as well as byRituxan®, as indicated in the figure. Blue bars: live cells; maroon barson the right of each pair: Annexin+/PI+.

FIG. 52 provides a graphic depiction of the direct cell killing ofvarious CD20-binding scorpions and SMIPs, as well as by Rituxan®, asindicated in the figure.

FIG. 53 provides graphs of the ADCC activity induced by variousCD20-binding scorpions and SMIPs, as indicated in the figure, as well asby Rituxan®.

FIG. 54 provides graphs of the CDC activity induced by variousCD20-binding scorpions and SMIPs, as indicated in the figure, as well asby Rituxan®.

FIG. 55 provides histograms showing the levels of C1q binding toCD20-binding scorpions bound to Ramos B-cells.

FIG. 56 provides scatter plots of FACS analyses showing the loss ofmitochondrial membrane potential attributable to CD20-binding scorpions(2Lm20-4×2Lm20-4 and 011×2Lm20-4) and Rituxan®, relative to controls(upper panel); histograms of the percentage of cells with disruptedmitochondrial membrane potential (disrupted MMP: black bars) are shownin the lower panel.

FIG. 57 provides histograms showing the relative lack of caspase 3activation by CD20-binding scorpions (2Lm20-4×2Lm20-4 and 011×2Lm20-4),Rituximab, CD95, and controls.

FIG. 58 provides a composite of four Western blot analyses ofPoly(ADP-ribose) Polymerase and caspases 3, 7, and 9 from B-cellsshowing little degradation of any of these proteins attributable toCD20-binding scorpions binding to the cells.

FIG. 59 is a gel electrophoretogram of B-cell chromosomal DNAs showingthe degree of fragmentation attributable to CD20-binding scorpionsbinding to the cells.

FIG. 60 is a gel electrophoretogram of immunoprecipitates obtained witheach of an anti-phosphotyrosine antibody and an anti-SYK antibody. Theimmunoprecipitates were obtained from lysates of B-cells contacted withCD20-binding scorpions, as indicated in the figure.

FIG. 61 provides combination index plots of CD20-binding scorpions incombination therapies with each of doxorubicin, vincristine andrapamycin.

DETAILED DESCRIPTION

The present invention provides compositions of relatively small peptideshaving at least two binding regions or domains, which may provide one ormore binding specificities, derived from variable binding domains ofimmunoglobulins, such as antibodies, disposed terminally relative to aneffector domain comprising at least part of an immunoglobulin constantregion (i.e., a source from which a constant sub-region, as definedherein, may be derived), as well as nucleic acids, vectors and hostcells involved in the recombinant production of such peptides andmethods of using the peptide compositions in a variety of diagnostic andtherapeutic applications, including the treatment of a disorder as wellas the amelioration of at least one symptom of such a disorder. Thepeptide compositions advantageously arrange a second binding domainC-terminal to the effector domain, an arrangement that unexpectedlyprovides sterically unhindered or less hindered binding by at least twobinding domains of the peptide, while retaining an effector function orfunctions of the centrally disposed effector domain.

The first and second binding domains of the multivalent peptidesaccording to the invention may be the same (i.e., have identical orsubstantially identical amino acid sequences and be monospecific) ordifferent (and be multispecific). Although different in terms of primarystructure, the first and second binding domains may recognize and bindto the same epitope of a target molecule and would therefore bemonospecific. In many instances, however, the binding domains willdiffer structurally and will bind to different binding sites, resultingin a multivalent, multispecific protein. Those different binding sitesmay exist on a single target molecule or on different target molecules.In the case of the two binding molecules recognizing different targetmolecules, those target molecules may exist, e.g., on or in the samestructure (e.g., the surface of the same cell), or those targetmolecules may exist on or in separate structures or locales. Forexample, a multispecific binding protein according to the invention mayhave binding domains that specifically bind to target molecule on thesurfaces of distinct cell types. Alternatively, one binding domain mayspecifically bind to a target on a cell surface and the other bindingdomain may specifically bind to a target not found associated with acell, such as an extracellular structural (matrix) protein or a free(e.g., soluble or stromal) protein.

The first and second binding domains are derived from one or moreregions of the same, or different, immunoglobulin protein structuressuch as antibody molecules. The first and/or second binding domain mayexhibit a sequence identical to the sequence of a region of animmunoglobulin, or may be a modification of such a sequence to provide,e.g., altered binding properties or altered stability. Suchmodifications are known in the art and include alterations in amino acidsequence that contribute directly to the altered property such asaltered binding, for example by leading to an altered secondary orhigher order structure for the peptide. Also contemplated are modifiedamino acid sequences resulting from the incorporation of non-nativeamino acids, such as non-native conventional amino acids, unconventionalamino acids and imino acids. In some embodiments, the altered sequenceresults in altered post-translational processing, for example leading toan altered glycosylation pattern.

Any of a wide variety of binding domains derived from an immunoglobulinor immunoglobulin-like polypeptide (e.g., receptor) are contemplated foruse in scorpions. Binding domains derived from antibodies comprise theCDR regions of a V_(L) and a V_(H) domain, seen, e.g., in the context ofusing a binding domain from a humanized antibody. Binding domainscomprising complete V_(L) and V_(H) domains derived from an antibody maybe organized in either orientation. A scorpion according to theinvention may have any of the binding domains herein described. Forscorpions having at least one binding domain recognizing a B-cell,exemplary scorpions have at least one binding domain derived from CD3,CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD72,CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a/b, CD80, CD81, CD82, CD83,CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDw130, CD138 or CDw150. Insome embodiments, the scorpion is a multivalent binding proteincomprising at least one binding domain having a sequence selected fromthe group consisting of SEQ ID NOS: 2, 4, 6, 103, 105, 107 and 109. Insome embodiments, a scorpion comprises a binding domain comprising asequence selected from the group consisting of any of SEQ ID NOS:332-345. In some embodiments, a scorpion comprises a binding domaincomprising a sequence derived from immunoglobulin V_(L) and V_(H)domains, wherein the sequence is selected from the group consisting ofany of SEQ ID NOS: 355-365. The invention further contemplates scorpionscomprising a binding domain that has the opposite orientation of V_(L)and V_(H) having sequences deducible from any of SEQ ID NOS:355-365.

For embodiments in which either, or both, of the binding domains arederived from more than one region of an immunoglobulin (e.g., an IgV_(L) region and an Ig V_(H) region), the plurality of regions may bejoined by a linker peptide. Moreover, a linker may be used to join thefirst binding domain to a constant sub-region. Joinder of the constantsub-region to a second binding domain (i.e., binding domain 2 disposedtowards the C-terminus of a scorpion) is accomplished by a scorpionlinker. These scorpion linkers are preferably between about 2-45 aminoacids, or 2-38 amino acids, or 5-45 amino acids. For example, the H1linker is 2 amino acids in length and the STD2 linker is 38 amino acidsin length. Beyond general length considerations, a scorpion linkerregion suitable for use in the scorpions according to the inventionincludes an antibody hinge region selected from the group consisting ofIgG, IgA, IgD and IgE hinges and variants thereof. For example, thescorpion linker may be an antibody hinge region selected from the groupconsisting of human IgG1, human IgG2, human IgG3, and human IgG4, andvariants thereof. In some embodiments, the scorpion linker region has asingle cysteine residue for formation of an interchain disulfide bond.In other embodiments, the scorpion linker has two cysteine residues forformation of interchain disulfide bonds. In some embodiments, a scorpionlinker region is derived from an immunoglobulin hinge region or aC-lectin stalk region and comprises a sequence selected from the groupconsisting of SEQ ID NOS:111, 113, 115, 117, 119, 121, 123, 125, 127,129, 131, 133, 135, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167,169, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255,257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 287,289, 297, 305, 307, 309, 310, 311, 313, 314, 315, 316, 317, 318, 319,320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 346, 351,352, 353, 354, 373, 374, 375, 376 and 377. More generally, any sequenceof amino acids identified in the sequence listing as providing asequence derived from a hinge region is contemplated for use as ascorpion linker in the scorpion molecules according to the invention. Inaddition, a scorpion linker derived from an Ig hinge is a hinge-likepeptide domain having at least one free cysteine capable ofparticipating in an interchain disulfide bond. Preferably, a scorpionlinker derived from an Ig hinge peptide retains a cysteine thatcorresponds to the hinge cysteine disposed towards the N-terminus ofthat hinge. Preferably, a scorpion linker derived from an IgG1 hinge hasone cysteine or has two cysteines corresponding to hinge cysteines.Additionally, a scorpion linker is a stalk region of a Type II C-lectinmolecule. In some embodiments, a scorpion comprises a scorpion linkerhaving a sequence selected from the group consisting of SEQ IDNOS:373-377.

The centrally disposed constant sub-region is derived from a constantregion of an immunoglobulin protein. The constant sub-region generallyis derived from a C_(H2) portion of a C_(H) region of an immunoglobulinin the abstract, although it may be derived from a C_(H2)-C_(H3)portion. Optionally, the constant sub-region may be derived from ahinge-C_(H2) or hinge-C_(H2)-C_(H3) portion of an immunoglobulin,placing a peptide corresponding to an Ig hinge region N-terminal to theconstant sub-region and disposed between the constant sub-region andbinding domain 1. Also, portions of the constant sub-region may bederived from the C_(H) regions of different immunoglobulins. Further,the peptide corresponding to an Ig CH3 may be truncated, leaving aC-terminal amino acid sequence selected from the group consisting of SEQID NOS:366-371. It is preferred, however, that in embodiments in which ascorpion hinge is a hinge-like peptide derived from an immunoglobulinhinge, that the scorpion linker and the constant sub-region be derivedfrom the same type of immunoglobulin. The constant sub-region providesat least one activity associated with a C_(H) region of animmunoglobulin, such as antibody-dependent cell-mediated cytotoxicity(ADCC), complement-dependent cytotoxicity (CDC), protein A binding,binding to at least one F_(C) receptor, reproducibly detectablestability relative to a protein according to the invention except forthe absence of a constant sub-region, and perhaps placental transferwhere generational transfer of a molecule according to the inventionwould be advantageous, as recognized by one of skill in the art. As withthe above-described binding domains, the constant sub-region is derivedfrom at least one immunoglobulin molecule and exhibits an identical orsubstantially identical amino acid sequence to a region or regions of atleast one immunoglobulin. In some embodiments, the constant sub-regionis modified from the sequence or sequences of at least oneimmunoglobulin (by substitution of one or more non-native conventionalor unconventional, e.g., synthetic, amino acids or imino acids),resulting in a primary structure that may yield an altered secondary orhigher order structure with altered properties associated therewith, ormay lead to alterations in post-translational processing, such asglycosylation.

For those binding domains and constant sub-regions exhibiting anidentical or substantially identical amino acid sequence to one or moreimmunoglobulin polypeptides, the post-translational modifications of themolecule according to the invention may result in a molecule modifiedrelative to the immunoglobulin(s) serving as a basis for modification.For example, using techniques known in the art, a host cell may bemodified, e.g. a CHO cell, in a manner that leads to an alteredpolypeptide glycosylation pattern relative to that polypeptide in anunmodified (e.g., CHO) host cell.

Provided with such molecules, and the methods of recombinantly producingthem in vivo, new avenues of targeted diagnostics and therapeutics havebeen opened to allow, e.g., for the targeted recruitment of effectorcells of the immune system (e.g., cytotoxic T lymphocytes, naturalkiller cells, and the like) to cells, tissues, agents and foreignobjects to be destroyed or sequestered, such as cancer cells andinfectious agents. In addition to localizing therapeutic cells to a siteof treatment, the peptides are useful in localizing therapeuticcompounds, such as radiolabeled proteins. Further, the peptides are alsouseful in scavenging deleterious compositions, for example byassociating a deleterious composition, such as a toxin, with a cellcapable of destroying or eliminating that toxin (e.g., a macrophage).The molecules of the invention are useful in modulating the activity ofbinding partner molecules, such as cell surface receptors. This is shownin FIG. 17 where apoptotic signaling through CD20 and/or CD37 ismarkedly enhanced by a molecule of the present invention. The effect ofthis signaling is the death of the targeted cell. Diseases andconditions where the elimination of defined cell populations isbeneficial would include infectious and parasitic diseases, inflammatoryand autoimmune conditions, malignancies, and the like. One skilled inthe art would recognize that there is no limitation of the approach tothe enhancement of apoptotic signaling. Mitotic signaling and signalingleading to differentiation, activation, or inactivation of defined cellpopulations can be induced by molecules of the present invention throughthe appropriate selection of binding partner molecules. Furtherconsideration of the disclosure of the invention will be facilitated bya consideration of the following express definitions of terms usedherein.

A “single-chain binding protein” is a single contiguous arrangement ofcovalently linked amino acids, with the chain capable of specificallybinding to one or more binding partners sharing sufficient determinantsof a binding site to be detectably bound by the single-chain bindingprotein. Exemplary binding partners include proteins, carbohydrates,lipids and small molecules.

For ease of exposition, “derivatives” and “variants” of proteins,polypeptides, and peptides according to the invention are described interms of differences from proteins and/or polypeptides and/or peptidesaccording to the invention, meaning that the derivatives and variants,which are proteins/polypeptides/peptides according to the invention,differ from underivatized or non-variant proteins, polypeptides orpeptides of the invention in the manner defined. One of skill in the artwould understand that the derivatives and variants themselves areproteins, polypeptides and peptides according to the invention.

An “antibody” is given the broadest definition consistent with itsmeaning in the art, and includes proteins, polypeptides and peptidescapable of binding to at least one binding partner, such as aproteinaceous or non-proteinaceous antigen. An “antibody” as used hereinincludes members of the immunoglobulin superfamily of proteins, of anyspecies, of single- or multiple-chain composition, and variants,analogs, derivatives and fragments of such molecules. Specifically, an“antibody” includes any form of antibody known in the art, including butnot limited to, monoclonal and polyclonal antibodies, chimericantibodies, CDR-grafted antibodies, humanized antibodies, single-chainvariable fragments, bi-specific antibodies, diabodies, antibody fusions,and the like.

A “binding domain” is a peptide region, such as a fragment of apolypeptide derived from an immunoglobulin (e.g., an antibody), thatspecifically binds one or more specific binding partners. If a pluralityof binding partners exists, those partners share binding determinantssufficient to detectably bind to the binding domain. Preferably, thebinding domain is a contiguous sequence of amino acids.

An “epitope” is given its ordinary meaning herein of a single antigenicsite, i.e., an antigenic determinant, on a substance (e.g., a protein)with which an antibody specifically interacts, for example by binding.Other terms that have acquired well-settled meanings in theimmunoglobulin (e.g., antibody) art, such as a “variable light region,”variable heavy region,” “constant light region,” constant heavy region,”“antibody hinge region,” “complementarity determining region,”“framework region,” “antibody isotype,” “F_(C) region,” “single-chainvariable fragment” or “scFv,” “diabody,” “chimera,” “CDR-graftedantibody,” “humanized antibody,” “shaped antibody,” “antibody fusion,”and the like, are each given those well-settled meanings known in theart, unless otherwise expressly noted herein.

Terms understood by those in the art as referring to antibody technologyare each given the meaning acquired in the art, unless expressly definedherein. Examples of such terms are “V_(L)” and “V_(H)”, referring to thevariable binding region derived from an antibody light and heavy chain,respectively; and C_(L) and C_(H), referring to an “immunoglobulinconstant region,” i.e., a constant region derived from an antibody lightor heavy chain, respectively, with the latter region understood to befurther divisible into C_(H1), C_(H2), C_(H3) and C_(H4) constant regiondomains, depending on the antibody isotype (IgA, IgD, IgE, IgG, IgM)from which the region was derived. CDR means “complementaritydetermining region.” A “hinge region” is derived from the amino acidsequence interposed between, and connecting, the C_(H1) and C_(H2)regions of a single chain of an antibody, which is known in the art asproviding flexibility, in the form of a “hinge,” to whole antibodies.

A “constant sub-region” is a term defined herein to refer to a peptide,polypeptide, or protein sequence that corresponds to, or is derivedfrom, one or more constant region domains of an antibody. Thus, aconstant sub-region may include any or all of the following domains: aC_(H1) domain, a hinge region, a C_(H2) domain, a C_(H3) domain (IgA,IgD, IgG, IgE, and IgM), and a C_(H4) domain (IgE, IgM). A constantsub-region as defined herein, therefore, can refer to a polypeptideregion corresponding to an entire constant region of an antibody, or aportion thereof. Typically, a constant sub-region of a polypeptide, orencoding nucleic acid, of the invention has a hinge, C_(H2) domain, andC_(H3) domain.

An “effector function” is a function associated with or provided by aconstant region of an antibody. Exemplary effector functions includeantibody-dependent cell-mediated cytotoxicity (ADCC), complementactivation and complement-dependent cytotoxicity (CDC), F_(C) receptorbinding, and increased plasma half-life, as well as placental transfer.An effector function of a composition according to the invention isdetectable; preferably, the specific activity of the compositionaccording to the invention for that function is about the same as thespecific activity of a wild-type antibody with respect to that effectorfunction, i.e., the constant sub-region of the multivalent bindingmolecule preferably has not lost any effector function relative to awild-type antibody]

A “linker” is a peptide, or polynucleotide, that joins or links otherpeptides or polynucleotides. Typically, a peptide linker is anoligopeptide of from about 2-50 amino acids, with typical polynucleotidelinkers encoding such a peptide linker and, thus, being about 6-150nucleotides in length. Linkers join the first binding domain to aconstant sub-region domain. An exemplary peptide linker is (Gly₄Ser)₃. Ascorpion linker is used to join the C-terminal end of a constantsub-region to a second binding domain. The scorpion linker may bederived from an immunoglobulin hinge region or from the stalk region ofa type II C-lectin, as described in greater detail below.

A “target” is given more than one meaning, with the context of usagedefining an unambiguous meaning in each instance. In its narrowestsense, a “target” is a binding site, i.e., the binding domain of abinding partner for a peptide composition according to the invention. Ina broader sense, “target” or “molecular target” refers to the entirebinding partner (e.g., a protein), which necessarily exhibits thebinding site. Specific targets, such as “CD20,” “CD37,” and the like,are each given the ordinary meaning the term has acquired in the art. A“target cell” is any prokaryotic or eukaryotic cell, whether healthy ordiseased, that is associated with a target molecule according to theinvention. Of course, target molecules are also found unassociated withany cell (i.e., a cell-free target) or in association with othercompositions such as viruses (including bacteriophage), organic orinorganic target molecule carriers, and foreign objects.

Examples of materials with which a target molecule may be associatedinclude autologous cells (e.g., cancer cells or other diseased cells),infectious agents (e.g., infectious cells and infectious viruses), andthe like. A target molecule may be associated with an enucleated cell, acell membrane, a liposome, a sponge, a gel, a capsule, a tablet, and thelike, which may be used to deliver, transport or localize a targetmolecule, regardless of intended use (e.g., for medical treatment, as aresult of benign or unintentional provision, or to further abioterrorist threat). “Cell-free,” “virus-free,” “carrier-free,”“object-free,” and the like refer to target molecules that are notassociated with the specified composition or material.

“Binding affinity” refers to the strength of non-covalent binding of thepeptide compositions of the invention and their binding partners.Preferably, binding affinity refers to a quantitative measure of theattraction between members of a binding pair.

An “adjuvant” is a substance that increases or aids the functionaleffect of a compound with which it is in association, such as in theform of a pharmaceutical composition comprising an active agent and anadjuvant. An “excipient” is an inert substance used as a diluent informulating a pharmaceutical composition. A “carrier” is a typicallyinert substance used to provide a vehicle for delivering apharmaceutical composition.

“Host cell” refers to any cell, prokaryotic or eukaryotic, in which isfound a polynucleotide, protein or peptide according to the invention.

“Introducing” a nucleic acid or polynucleotide into a host cell meansproviding for entry of the nucleic acid or polynucleotide into that cellby any means known in the art, including but not limited to, in vitrosalt-mediated precipitations and other forms of transformation of nakednucleic acid/polynucleotide or vector-borne nucleic acid/polynucleotide,virus-mediated infection and optionally transduction, with or without a“helper” molecule, ballistic projectile delivery, conjugation, and thelike.

“Incubating” a host cell means maintaining that cell under environmentalconditions known in the art to be suitable for a given purpose, such asgene expression. Such conditions, including temperature, ionic strength,oxygen tension, carbon dioxide concentration, nutrient composition, andthe like, are well known in the art.

“Isolating” a compound, such as a protein or peptide according to theinvention, means separating that compound from at least one distinctcompound with which it is found associated in nature, such as in a hostcell expressing the compound to be isolated, e.g. by isolating spentculture medium containing the compound from the host cells grown in thatmedium.

An “organism in need” is any organism at risk of, or suffering from, anydisease, disorder or condition that is amenable to treatment oramelioration with a composition according to the invention, includingbut not limited to any of various forms of cancer, any of a number ofautoimmune diseases, radiation poisoning due to radiolabeled proteins,peptides and like compounds, ingested or internally produced toxins, andthe like, as will become apparent upon review of the entire disclosure.Preferably, an organism in need is a human patient.

“Ameliorating” a symptom of a disease means detectably reducing theseverity of that symptom of disease, as would be known in the art.Exemplary symptoms include pain, heat, swelling and joint stiffness.

Unless clear from context, the terms “protein,” “peptide,” and“polypeptide” are used interchangeably herein, with each referring to atleast one contiguous chain of amino acids. Analogously, the terms“polynucleotide,” “nucleic acid,” and “nucleic acid molecule” are usedinterchangeably unless it is clear from context that a particular, andnon-interchangeable, meaning is intended.

“Pharmaceutically acceptable salt” refers to salts of the compounds ofthe present invention derived from the combination of such compounds andan organic or inorganic acid (acid addition salts) or an organic orinorganic base (base addition salts).

Using the terms as defined above, a general description of the variousaspects of the invention is provided below. Following the generaldescription, working examples are presented to provide supplementaryevidence of the operability and usefulness of the invention disclosedherein.

Proteins and Polypeptides

In certain embodiments of the invention, there are provided any of theherein-described multivalent binding proteins with effector function,including binding domain-immunoglobulin fusion proteins, wherein themultivalent binding protein or peptide with effector function comprisestwo or more binding domain polypeptide sequences. Each of the bindingdomain polypeptide sequences is capable of binding or specificallybinding to a target(s), such as an antigen(s), which target(s) orantigen(s) may be the same or may be different. The binding domainpolypeptide sequence may be derived from an antigen variable region orit may be derived from immunoglobulin-like molecules, e.g., receptorsthat fold in ways that mimic immunoglobulin molecules. The antibodiesfrom which the binding domains are derived may be antibodies that arepolyclonal, including monospecific polyclonal, monoclonal (mAbs),recombinant, chimeric, humanized (such as CDR-grafted), human,single-chain, catalytic, and any other form of antibody known in theart, as well as fragments, variants or derivatives thereof. In someembodiments, each of the binding domains of the protein according to theinvention is derived from a complete variable region of animmunoglobulin. In preferred embodiments, the binding domains are eachbased on a human Ig variable region. In other embodiments, the proteinis derived from a fragment of an Ig variable region. In suchembodiments, it is preferred that each binding domain polypeptidesequence correspond to the sequences of each of the complementaritydetermining regions of a given Ig variable region. Also contemplatedwithin the invention are binding domains that correspond to fewer thanall CDRs of a given Ig variable region, provided that such bindingdomains retain the capacity to specifically bind to at least one target.

The multivalent binding protein with effector function also has aconstant sub-region sequence derived from an immunoglobulin constantregion, preferably an antibody heavy chain constant region, covalentlyjuxtaposed between the two binding domains in the multivalent bindingprotein with effector function.

The multivalent binding protein with effector function also has ascorpion linker that joins the C-terminal end of the constant sub-regionto the N-terminal end of binding domain 2. The scorpion linker is not ahelical peptide and may be derived from an antibody hinge region, from aregion connecting binding domains of an immunoglobulin, or from thestalk region of type II C-lectins. The scorpion linker may be derivedfrom a wild-type hinge region of an immunoglobulin, such as an IgG1,IgG2, IgG3, IgG4, IgA, IgD or an IgE hinge region. In other embodiments,the invention provides multivalent binding proteins with altered hinges.One category of altered hinge regions suitable for inclusion in themultivalent binding proteins is the category of hinges with an alterednumber of Cysteine residues, particularly those Cys residues known inthe art to be involved in interchain disulfide bond formation inimmunoglobulin counterpart molecules having wild-type hinges. Thus,proteins may have an IgG1 hinge in which one of the three Cys residuescapable of participating in interchain disulfide bond formations ismissing. To indicate the Cysteine sub-structure of altered hinges, theCys subsequence is presented from N- to C-terminus. Using thisidentification system, the multivalent binding proteins with altered IgGhinges include hinge structures characterized as cxc, xxc, ccx, xxc,xcx, cxx, and xxx. The Cys residue may be either deleted or substitutedby an amino acid that results in a conservative substitution or anon-conservative substitution. In some embodiments, the Cysteine isreplaced by a Serine. For proteins with scorpion linkers comprising IgG1hinges, the number of cysteines corresponding to hinge cysteines isreduced to 1 or 2, preferably with one of those cysteines correspondingto the hinge cysteine disposed closest to the N-terminus of the hinge.

For proteins with scorpion linkers comprising IgG2 hinges, there may be0, 1, 2, 3, or 4 Cys residues. For scorpion linkers comprising alteredIgG2 hinges containing 1, 2 or 3 Cys residues, all possible subsets ofCys residues are contemplated. Thus, for such linkers having one Cys,the multivalent binding proteins may have the following Cys motif in thehinge region: cxxx, xcxx, xxcx, or xxxc. For scorpion linkers comprisingIgG2 hinge variants having 2 or 3 Cys residues, all possiblecombinations of retained and substituted (or deleted) Cys residues arecontemplated. For multivalent binding proteins with scorpion linkerscomprising altered IgG3 or altered IgG4 hinge regions, a reduction inCys residues from 1 to one less than the complete number of Cys residuesin the hinge region is contemplated, regardless of whether the loss isthrough deletion or substitution by conservative or non-conservativeamino acids (e.g., Serine). In like manner, multivalent binding proteinshaving a scorpion linker comprising a wild-type IgA, IgD or IgE hingeare contemplated, as are corresponding altered hinge regions having areduced number of Cys residues extending from 0 to one less than thetotal number of Cys residues found in the corresponding wild-type hinge.In some embodiments having an IgG1 hinge, the first, or N-terminal, Cysresidue of the hinge is retained. For proteins with either wild-type oraltered hinge regions, it is contemplated that the multivalent bindingproteins will be single-chain molecules capable of forminghomo-multimers, such as dimers, e.g., by disulfide bond formation.Further, proteins with altered hinges may have alterations at thetermini of the hinge region, e.g., loss or substitution of one or moreamino acid residues at the N-terminus, C-terminus or both termini of agiven region or domain, such as a hinge domain, as disclosed herein.

In another exemplary embodiment, the constant sub-region is derived froma constant region that comprises a native, or an engineered, IgD hingeregion. The wild-type human IgD hinge has one cysteine that forms adisulfide bond with the light chain in the native IgD structure. In someembodiments, this IgD hinge cysteine is mutated (e.g., deleted) togenerate an altered hinge for use as a connecting region between bindingdomains of, for example, a bispecific molecule. Other amino acid changesor deletions or alterations in an IgD hinge that do not result inundesired hinge inflexibility are within the scope of the invention.Native or engineered IgD hinge regions from other species are alsowithin the scope of the invention, as are humanized native or engineeredIgD hinges from non-human species, and (other non IgD) hinge regionsfrom other human, or non-human, antibody isotypes, (such as the llamaIgG2 hinge).

The invention further comprehends constant sub-regions attached toscorpion linkers that may be derived from hinges that correspond to aknown hinge region, such as an IgG1 hinge or an IgD hinge, as notedabove. The constant sub-region may contain a modified or altered(relative to wild-type) hinge region in which at least one cysteineresidue known to participate in inter-chain disulfide bond linkage isreplaced by another amino acid in a conservative substitution (e.g., Serfor Cys) or a non-conservative substitution. The constant sub-regiondoes not include a peptide region or domain that corresponds to animmunoglobulin C_(H1) domain.

Alternative hinge and linker sequences that can be used as connectingregions are from portions of cell surface receptors that connectimmunoglobulin V-like or immunoglobulin C-like domains. Regions betweenIg V-like domains where the cell surface receptor contains multiple IgV-like domains in tandem, and between Ig C-like domains where the cellsurface receptor contains multiple tandem Ig C-like regions are alsocontemplated as connecting regions. Hinge and linker sequences aretypically from 5 to 60 amino acids long, and may be primarily flexible,but may also provide more rigid characteristics. In addition, linkersfrequently provide spacing that facilitates minimization of sterichindrance between the binding domains. Preferably, these hinge andlinker peptides are primarily a helical in structure, with minimal βsheet structure. The preferred sequences are stable in plasma and serumand are resistant to proteolytic cleavage. The preferred sequences maycontain a naturally occurring or added motif such as the CPPC motif thatconfers a disulfide bond to stabilize dimer formation. The preferredsequences may contain one or more glycosylation sites. Examples ofpreferred hinge and linker sequences include, but are not limited to,the interdomain regions between the Ig V-like and Ig C-like regions ofCD2, CD4, CD22, CD33, CD48, CD58, CD66, CD80, CD86, CD150, CD166, andCD244.

The constant sub-region may be derived from a camelid constant region,such as either a llama or camel IgG2 or IgG3.

Specifically contemplated is a constant sub-region having theC_(H2)-C_(H3) region from any Ig class, or from any IgG subclass, suchas IgG1 (e.g., human IgG1). In preferred embodiments, the constantsub-region and the scorpion linker derived from an immunoglobulin hingeare both derived from the same Ig class. In other preferred embodiments,the constant sub-region and the scorpion linker derived from animmunoglobulin hinge are both derived from the same Ig sub-class. Theconstant sub-region also may be a CH3 domain from any Ig class orsubclass, such as IgG1 (e.g., human IgG1), provided that it isassociated with at least one immunoglobulin effector function.

The constant sub-region does not correspond to a complete immunoglobulinconstant region (i.e., C_(H1)-hinge-C_(H2)-C_(H3)) of the IgG class. Theconstant sub-region may correspond to a complete immunoglobulin constantregion of other classes. IgA constant domains, such as an IgA1 hinge, anIgA2 hinge, an IgA C_(H2) and an IgA C_(H3) domains with a mutated ormissing tailpiece are also contemplated as constant sub-regions.Further, any light chain constant domain may function as a constantsub-region, e.g., C_(K) or any C_(L). The constant sub-region may alsoinclude JH or JK, with or without a hinge. The constant sub-region mayalso correspond to engineered antibodies in which, e.g., a loop grafthas been constructed by making selected amino acid substitutions usingan IgG framework to generate a binding site for a receptor other than anatural F_(C)R (CD16, CD32, CD64, F_(C)εR1), as would be understood inthe art. An exemplary constant sub-region of this type is an IgGC_(H2)-C_(H3) region modified to have a CD89 binding site.

This aspect of the invention provides a multivalent binding protein orpeptide having effector function, comprising, consisting essentially of,or consisting of (a) an N-terminally disposed binding domain polypeptidesequence derived from an immunoglobulin that is fused or otherwiseconnected to (b) a constant sub-region polypeptide sequence derived froman immunoglobulin constant region, which preferably includes a hingeregion sequence, wherein the hinge region polypeptide may be asdescribed herein, and may comprise, consist essentially of, or consistof, for example, an alternative hinge region polypeptide sequence, inturn fused or otherwise connected to (c) a C-terminally disposed secondnative or engineered binding domain polypeptide sequence derived from animmunoglobulin.

The centrally disposed constant sub-region polypeptide sequence derivedfrom an immunoglobulin constant region is capable of at least oneimmunological activity selected from the group consisting of antibodydependent cell-mediated cytotoxicity, CDC, complement fixation, andF_(C) receptor binding, and the binding domain polypeptides are eachcapable of binding or specifically binding to a target, such as anantigen, wherein the targets may be the same or different, and may befound in effectively the same physiological environment (e.g., thesurface of the same cell) or in different environments (e.g., differentcell surfaces, a cell surface and a cell-free location, such as insolution).

This aspect of the invention also comprehends variant proteins orpolypeptides exhibiting an effector function that are at least 80%, andpreferably 85%, 90%, 95% or 99% identical to a multivalent protein witheffector function of specific sequence as disclosed herein.

Polynucleotides

The invention also provides polynucleotides (isolated or purified orpure polynucleotides) encoding the proteins or peptides according to theinvention, vectors (including cloning vectors and expression vectors)comprising such polynucleotides, and cells (e.g., host cells)transformed or transfected with a polynucleotide or vector according tothe invention. In encoding the proteins or polypeptides of theinvention, the polynucleotides encode a first binding domain, a secondbinding domain and an F_(C) domain, all derived from immunoglobulins,preferably human immunoglobulins. Each binding domain may contain asequence corresponding to a full-length variable region sequence (eitherheavy chain and/or light chain), or to a partial sequence thereof,provided that each such binding domain retains the capacity tospecifically bind. The F_(C) domain may have a sequence that correspondsto a full-length immunoglobulin F_(C) domain sequence or to a partialsequence thereof, provided that the F_(C) domain exhibits at least oneeffector function as defined herein. In addition, each of the bindingdomains may be joined to the F_(C) domain via a linker peptide thattypically is at least 8, and preferably at least 13, amino acids inlength. A preferred linker sequence is a sequence based on the Gly₄Sermotif, such as (Gly₄Ser)₃.

Variants of the multivalent binding protein with effector function arealso comprehended by the invention. Variant polynucleotides are at least90%, and preferably 95%, 99%, or 99.9% identical to one of thepolynucleotides of defined sequence as described herein, or thathybridizes to one of those polynucleotides of defined sequence understringent hybridization conditions of 0.015 M sodium chloride, 0.0015 Msodium citrate at 65-68° C. or 0.015 M sodium chloride, 0.0015M sodiumcitrate, and 50% formamide at 42° C. The polynucleotide variants retainthe capacity to encode a multivalent binding protein with effectorfunction.

The term “stringent” is used to refer to conditions that are commonlyunderstood in the art as stringent. Hybridization stringency isprincipally determined by temperature, ionic strength, and theconcentration of denaturing agents such as formamide. Examples ofstringent conditions for hybridization and washing are 0.015 M sodiumchloride, 0.0015 M sodium citrate at 65-68° C. or 0.015 M sodiumchloride, 0.0015M sodium citrate, and 50% formamide at 42° C. SeeSambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., ColdSpring Harbor Laboratory, (Cold Spring Harbor, N.Y. 1989).

More stringent conditions (such as higher temperature, lower ionicstrength, higher formamide, or other denaturing agent) may also be used;however, the rate of hybridization will be affected. In instanceswherein hybridization of deoxyoligonucleotides is concerned, additionalexemplary stringent hybridization conditions include washing in 6×SSC,0.05% sodium pyrophosphate at 37° C. (for 14-base oligonucleotides), 48°C. (for 17-base oligonucleotides), 55° C. (for 20-baseoligonucleotides), and 60° C. (for 23-base oligonucleotides).

In a related aspect of the invention, there is provided a method ofproducing a polypeptide or protein or other construct of the invention,for example, including a multivalent binding protein or peptide havingeffector function, comprising the steps of (a) culturing a host cell asdescribed or provided for herein under conditions that permit expressionof the construct; and (b) isolating the expression product, for example,the multivalent binding protein or peptide with effector function fromthe host cell or host cell culture.

Constructs

The present invention also relates to vectors, and to constructsprepared from known vectors, that each include a polynucleotide ornucleic acid of the invention, and in particular to recombinantexpression constructs, including any of various known constructs,including delivery constructs, useful for gene therapy, that include anynucleic acids encoding multivalent, for example, multispecific,including bi-specific, binding proteins and polypeptides with effectorfunction, as provided herein; to host cells which are geneticallyengineered with vectors and/or other constructs of the invention and tomethods of administering expression or other constructs comprisingnucleic acid sequences encoding multivalent, for example, multispecific,including bi-specific, binding proteins with effector function, orfragments or variants thereof, by recombinant techniques.

Various constructs of the invention including multivalent, for example,multispecific binding proteins with effector function, can be expressedin virtually any host cell, including in vivo host cells in the case ofuse for gene therapy, under the control of appropriate promoters,depending on the nature of the construct (e.g., type of promoter, asdescribed above), and on the nature of the desired host cell (e.g.,postmitotic terminally differentiated or actively dividing; e.g.,maintenance of an expressible construct as an episome or integrated intothe host cell genome).

Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described, for example, in Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989). Exemplary cloning/expression vectors include, butare not limited to, cloning vectors, shuttle vectors, and expressionconstructs, that may be based on plasmids, phagemids, phasmids, cosmids,viruses, artificial chromosomes, or any nucleic acid vehicle suitablefor amplification, transfer, and/or expression of a polynucleotidecontained therein that is known in the art. As noted herein, inpreferred embodiments of the invention, recombinant expression isconducted in mammalian cells that have been transfected, transformed ortransduced with a nucleic acid according to the invention. See also, forexample, Machida, C A., “Viral Vectors for Gene Therapy: Methods andProtocols”; Wolff, J A, “Gene Therapeutics: Methods and Applications ofDirect Gene Transfer” (Birkhauser 1994); Stein, U and Walther, W (eds.,“Gene Therapy of Cancer: Methods and Protocols” (Humana Press 2000);Robbins, P D (ed.), “Gene Therapy Protocols” (Humana Press 1997);Morgan, J R (ed.), “Gene Therapy Protocols” (Humana Press 2002); Meager,A (ed.), “Gene Therapy Technologies, Applications and Regulations: FromLaboratory to Clinic” (John Wiley & Sons Inc. 1999); MacHida, C A andConstant, J G, “Viral Vectors for Gene Therapy: Methods and Protocols”(Humana Press 2002); “New Methods Of Gene Therapy For Genetic MetabolicDiseases NIH Guide,” Volume 22, Number 35, Oct. 1, 1993. See also U.S.Pat. Nos. 6,384,210; 6,384,203; 6,384,202; 6,384,018; 6,383,814;6,383,811; 6,383,795; 6,383,794; 6,383,785; 6,383,753; 6,383,746;6,383,743; 6,383,738; 6,383,737; 6,383,733; 6,383,522; 6,383,512;6,383,481; 6,383,478; 6,383,138; 6,380,382; 6,380,371; 6,380,369;6,380,362; 6,380,170; 6,380,169; 6,379,967; and 6,379,966.

Typically, expression constructs are derived from plasmid vectors. Onepreferred construct is a modified pNASS vector (Clontech, Palo Alto,Calif.), which has nucleic acid sequences encoding an ampicillinresistance gene, a polyadenylation signal and a T7 promoter site. Othersuitable mammalian expression vectors are well known (see, e.g., Ausubelet al., 1995; Sambrook et al., supra; see also, e.g., catalogues fromInvitrogen, San Diego, Calif.; Novagen, Madison, Wis.; Pharmacia,Piscataway, N.J.). Presently preferred constructs may be prepared thatinclude a dihydrofolate reductase (DHFR)-encoding sequence undersuitable regulatory control, for promoting enhanced production levels ofthe multivalent binding protein with effector function, which levelsresult from gene amplification following application of an appropriateselection agent (e.g., methotrexate).

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence, as described above. Avector in operable linkage with a polynucleotide according to theinvention yields a cloning or expression construct. Exemplarycloning/expression constructs contain at least one expression controlelement, e.g., a promoter, operably linked to a polynucleotide of theinvention. Additional expression control elements, such as enhancers,factor-specific binding sites, terminators, and ribosome binding sitesare also contemplated in the vectors and cloning/expression constructsaccording to the invention. The heterologous structural sequence of thepolynucleotide according to the invention is assembled in appropriatephase with translation initiation and termination sequences. Thus, forexample, the multivalent binding protein-encoding nucleic acids asprovided herein may be included in any one of a variety of expressionvector constructs as a recombinant expression construct for expressingsuch a protein in a host cell. In certain preferred embodiments theconstructs, are included in formulations that are administered in vivo.Such vectors and constructs include chromosomal, nonchromosomal andsynthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids;phage DNA; yeast plasmids; vectors derived from combinations of plasmidsand phage DNA, viral DNA, such as vaccinia, adenovirus, fowl pox virus,and pseudorabies, or replication deficient retroviruses as describedbelow. However, any other vector may be used for preparation of arecombinant expression construct, and in preferred embodiments such avector will be replicable and viable in the host.

The appropriate DNA sequence(s) may be inserted into a vector, forexample, by a variety of procedures. In general, a DNA sequence isinserted into an appropriate restriction endonuclease cleavage site(s)by procedures known in the art. Standard techniques for cloning, DNAisolation, amplification and purification, for enzymatic reactionsinvolving DNA ligase, DNA polymerase, restriction endonucleases and thelike, and various separation techniques are contemplated. A number ofstandard techniques are described, for example, in Ausubel et al. (1993Current Protocols in Molecular Biology, Greene Publ. Assoc. Inc. & JohnWiley & Sons, Inc., Boston, Mass.); Sambrook et al. (1989 MolecularCloning, Second Ed., Cold Spring Harbor Laboratory, Plainview, N.Y.);Maniatis et al. (1982 Molecular Cloning, Cold Spring Harbor Laboratory,Plainview, N.Y.); Glover (Ed.) (1985 DNA Cloning Vol. I and II, IRLPress, Oxford, UK); Hames and Higgins (Eds.), (1985 Nucleic AcidHybridization, IRL Press, Oxford, UK); and elsewhere.

The DNA sequence in the expression vector is operatively linked to atleast one appropriate expression control sequence (e.g., a constitutivepromoter or a regulated promoter) to direct mRNA synthesis.Representative examples of such expression control sequences includepromoters of eukaryotic cells or their viruses, as described above.Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art, and preparation ofcertain particularly preferred recombinant expression constructscomprising at least one promoter or regulated promoter operably linkedto a nucleic acid encoding a protein or polypeptide according to theinvention is described herein.

Transcription of the DNA encoding proteins and polypeptides of theinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector. Examples include the SV40 enhancer on the lateside of the replication origin by 100 to 270, a cytomegalovirus earlypromoter enhancer, the polyoma enhancer on the late side of thereplication origin, and adenovirus enhancers.

Gene therapies using the nucleic acids of the invention are alsocontemplated, comprising strategies to replace defective genes or addnew genes to cells and/or tissues, and is being developed forapplication in the treatment of cancer, the correction of metabolicdisorders and in the field of immunotherapy. Gene therapies of theinvention include the use of various constructs of the invention, withor without a separate carrier or delivery vehicle or constructs, fortreatment of the diseases, disorders, and/or conditions noted herein.Such constructs may also be used as vaccines for treatment or preventionof the diseases, disorders, and/or conditions noted herein. DNAvaccines, for example, make use of polynucleotides encoding immunogenicprotein and nucleic acid determinants to stimulate the immune systemagainst pathogens or tumor cells. Such strategies can stimulate eitheracquired or innate immunity or can involve the modification of immunefunction through cytokine expression. In vivo gene therapy involves thedirect injection of genetic material into a patient or animal, typicallyto treat, prevent or ameliorate a disease or symptoms associated with adisease. Vaccines and immune modulation are systemic therapies. Withtissue-specific in vivo therapies, such as those that aim to treatcancer, localized gene delivery and/or expression/targeting systems arepreferred. Diverse gene therapy vectors that target specific tissues areknown in the art, and procedures have been developed to physicallytarget specific tissues, for example, using catheter-based technologies,all of which are contemplated herein.

Ex vivo approaches to gene therapy are also contemplated herein andinvolve the removal, genetic modification, expansion andre-administration of a subject's, e.g., human patient's, own cells.Examples include bone marrow transplantation for cancer treatment or thegenetic modification of lymphoid progenitor cells. Ex vivo gene therapyis preferably applied to the treatment of cells that are easilyaccessible and can survive in culture during the gene transfer process(such as blood or skin cells).

Useful gene therapy vectors include adenoviral vectors, lentiviralvectors, Adeno-associated virus (AAV) vectors, Herpes Simplex Virus(HSV) vectors, and retroviral vectors. Gene therapies may also becarried out using “naked DNA,” liposome-based delivery, lipid-baseddelivery (including DNA attached to positively charged lipids),electroporation, and ballistic projection.

In certain embodiments, including but not limited to gene therapyembodiments, the vector may be a viral vector such as, for example, aretroviral vector. Miller et al., 1989 Bio Techniques 7:980; Coffin andVarmus, 1996 Retroviruses, Cold Spring Harbor Laboratory Press, NY. Forexample, retroviruses from which the retroviral plasmid vectors may bederived include, but are not limited to, Moloney Murine Leukemia Virus,spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, HarveySarcoma virus, avian leukosis virus, gibbon ape leukemia virus, humanimmunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus,and mammary tumor virus.

Retroviruses are RNA viruses which can replicate and integrate into thegenome of a host cell via a DNA intermediate. This DNA intermediate, orprovirus, may be stably integrated into the host cell DNA. According tocertain embodiments of the present invention, an expression constructmay comprise a retrovirus into which a foreign gene that encodes aforeign protein is incorporated in place of normal retroviral RNA. Whenretroviral RNA enters a host cell coincident with infection, the foreigngene is also introduced into the cell, and may then be integrated intohost cell DNA as if it were part of the retroviral genome. Expression ofthis foreign gene within the host results in expression of the foreignprotein.

Most retroviral vector systems that have been developed for gene therapyare based on murine retroviruses. Such retroviruses exist in two forms,as free viral particles referred to as virions, or as provirusesintegrated into host cell DNA. The virion form of the virus contains thestructural and enzymatic proteins of the retrovirus (including theenzyme reverse transcriptase), two RNA copies of the viral genome, andportions of the source cell plasma membrane containing viral envelopeglycoprotein. The retroviral genome is organized into four main regions:the Long Terminal Repeat (LTR), which contains cis-acting elementsnecessary for the initiation and termination of transcription and issituated both 5′ and 3′ to the coding genes, and the three genesencoding gag, pol, and env. These three genes, gag, pol, and env,encode, respectively, internal viral structures, enzymatic proteins(such as integrase), and the envelope glycoprotein (designated gp70 andp15e) which confers infectivity and host range specificity of the virus,as well as the “R” peptide of undetermined function.

Separate packaging cell lines and vector-producing cell lines have beendeveloped because of safety concerns regarding the uses of retroviruses,including uses in expression constructs. Briefly, this methodologyemploys the use of two components, a retroviral vector and a packagingcell line (PCL). The retroviral vector contains long terminal repeats(LTRs), the foreign DNA to be transferred and a packaging sequence (y).This retroviral vector will not reproduce by itself because the geneswhich encode structural and envelope proteins are not included withinthe vector genome. The PCL contains genes encoding the gag, pol, and envproteins, but does not contain the packaging signal “y.” Thus, a PCL canonly form empty virion particles by itself. Within this general method,the retroviral vector is introduced into the PCL, thereby creating avector-producing cell line (VCL). This VCL manufactures virion particlescontaining only the foreign genome of the retroviral vector, andtherefore has previously been considered to be a safe retrovirus vectorfor therapeutic use.

A “retroviral vector construct” refers to an assembly which is, withinpreferred embodiments of the invention, capable of directing theexpression of a sequence(s) or gene(s) of interest, such as multivalentbinding protein-encoding nucleic acid sequences. Briefly, the retroviralvector construct must include a 5′ LTR, a tRNA binding site, a packagingsignal, an origin of second strand DNA synthesis and a 3′ LTR. A widevariety of heterologous sequences may be included within the vectorconstruct including, for example, sequences which encode a protein(e.g., cytotoxic protein, disease-associated antigen, immune accessorymolecule, or replacement gene), or which are useful as a molecule itself(e.g., as a ribozyme or antisense sequence).

Retroviral vector constructs of the present invention may be readilyconstructed from a wide variety of retroviruses, including for example,B, C, and D type retroviruses as well as spumaviruses and lentiviruses(see, e.g., RNA Tumor Viruses, Second Edition, Cold Spring HarborLaboratory, 1985). Such retroviruses may be readily obtained fromdepositories or collections such as the American Type Culture Collection(“ATCC”; Rockville, Md.), or isolated from known sources using commonlyavailable techniques. Any of the above retroviruses may be readilyutilized in order to assemble or construct retroviral vector constructs,packaging cells, or producer cells of the invention, given thedisclosure provided herein and standard recombinant techniques (e.g.,Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed., ColdSpring Harbor Laboratory Press, 1989; Kunkle, 1985 Proc. Natl. Acad.Sci. (USA) 82:488).

Suitable promoters for use in viral vectors generally may include, butare not limited to, the retroviral LTR; the SV40 promoter; and the humancytomegalovirus (CMV) promoter described in Miller, et al., 1989Biotechniques 7:980-990, or any other promoter (e.g., cellular promoterssuch as eukaryotic cellular promoters including, but not limited to, thehistone, pol III, and β-actin promoters). Other viral promoters that maybe employed include, but are not limited to, adenovirus promoters,thymidine kinase (TK) promoters, and B19 parvovirus promoters. Theselection of a suitable promoter will be apparent to those skilled inthe art from the teachings contained herein, and may be from amongeither regulated promoters or promoters as described above.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, ψ-2,ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy, 1:5-14(1990). The vector may transduce the packaging cells through any meansknown in the art. Such means include, but are not limited to,electroporation, the use of liposomes, and CaPO₄ precipitation. In onealternative, the retroviral plasmid vector may be encapsulated into aliposome, or coupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequence(s) encoding the multivalentbinding proteins with effector function. Such retroviral vectorparticles then may be employed to transduce eukaryotic cells, either invitro or in vivo. The transduced eukaryotic cells will express thenucleic acid sequence(s) encoding the protein or polypeptide. Eukaryoticcells that may be transduced include, but are not limited to, embryonicstem cells, as well as hematopoietic stem cells, hepatocytes,fibroblasts, circulating peripheral blood mononuclear andpolymorphonuclear cells including myelomonocytic cells, lymphocytes,myoblasts, tissue macrophages, dendritic cells, Kupffer cells, lymphoidand reticuloendothelial cells of the lymph nodes and spleen,keratinocytes, endothelial cells, and bronchial epithelial cells.

Host Cells

A further aspect of the invention provides a host cell transformed ortransfected with, or otherwise containing, any of the polynucleotides orcloning/expression constructs of the invention. The polynucleotides andcloning/expression constructs are introduced into suitable cells usingany method known in the art, including transformation, transfection andtransduction. Host cells include the cells of a subject undergoing exvivo cell therapy including, for example, ex vivo gene therapy.Eukaryotic host cells contemplated as an aspect of the invention whenharboring a polynucleotide, vector, or protein according to theinvention include, in addition to a subject's own cells (e.g., a humanpatient's own cells), VERO cells, HeLa cells, Chinese hamster ovary(CHO) cell lines (including modified CHO cells capable of modifying theglycosylation pattern of expressed multivalent binding molecules, seePublished US Patent Application No. 2003/0115614 A1), incorporatedherein by reference, COS cells (such as COS-7), W138, BHK, HepG2, 3T3,RIN, MDCK, A549, PC12, K562, HEK293 cells, HepG2 cells, N cells, 3T3cells, Spodoptera frugiperda cells (e.g., Sf9 cells), Saccharomycescerevisiae cells, and any other eukaryotic cell known in the art to beuseful in expressing, and optionally isolating, a protein or peptideaccording to the invention. Also contemplated are prokaryotic cells,including but not limited to, Escherichia coli, Bacillus subtilis,Salmonella typhimurium, a Streptomycete, or any prokaryotic cell knownin the art to be suitable for expressing, and optionally isolating, aprotein or peptide according to the invention. In isolating protein orpeptide from prokaryotic cells, in particular, it is contemplated thattechniques known in the art for extracting protein from inclusion bodiesmay be used. The selection of an appropriate host is within the scope ofthose skilled in the art from the teachings herein.

The engineered host cells can be cultured in a conventional nutrientmedium modified as appropriate for activating promoters, selectingtransformants, or amplifying particular genes. The culture conditionsfor particular host cells selected for expression, such as temperature,pH and the like, will be readily apparent to the ordinarily skilledartisan. Various mammalian cell culture systems can also be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzman, 1981 Cell 23:175, and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and, optionally, enhancer, and also anynecessary ribosome binding sites, polyadenylation site, splice donor andacceptor sites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences, for example as described herein regarding thepreparation of multivalent binding protein expression constructs. DNAsequences derived from the SV40 splice, and polyadenylation sites may beused to provide the required nontranscribed genetic elements.Introduction of the construct into the host cell can be effected by avariety of methods with which those skilled in the art will be familiar,including but not limited to, calcium phosphate transfection,DEAE-Dextran-mediated transfection, or electroporation (Davis et al.,1986 Basic Methods in Molecular Biology).

In one embodiment, a host cell is transduced by a recombinant viralconstruct directing the expression of a protein or polypeptide accordingto the invention. The transduced host cell produces viral particlescontaining expressed protein or polypeptide derived from portions of ahost cell membrane incorporated by the viral particles during viralbudding.

Pharmaceutical Compositions

In some embodiments, the compositions of the invention, such as amultivalent binding protein or a composition comprising a polynucleotideencoding such a protein as described herein, are suitable to beadministered under conditions and for a time sufficient to permitexpression of the encoded protein in a host cell in vivo or in vitro,for gene therapy, and the like. Such compositions may be formulated intopharmaceutical compositions for administration according to well knownmethodologies. Pharmaceutical compositions generally comprise one ormore recombinant expression constructs, and/or expression products ofsuch constructs, in combination with a pharmaceutically acceptablecarrier, excipient or diluent. Such carriers will be nontoxic torecipients at the dosages and concentrations employed. For nucleicacid-based formulations, or for formulations comprising expressionproducts according to the invention, about 0.01 μg/kg to about 100 mg/kgbody weight will be administered, for example, by the intradermal,subcutaneous, intramuscular or intravenous route, or by any route knownin the art to be suitable under a given set of circumstances. Apreferred dosage, for example, is about 1 μg/kg to about 1 mg/kg, withabout 5 μg/kg to about 200 μg/kg particularly preferred.

It will be evident to those skilled in the art that the number andfrequency of administration will be dependent upon the response of thehost. Pharmaceutically acceptable carriers for therapeutic use are wellknown in the pharmaceutical art, and are described, for example, inRemingtons Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaroedit. 1985). For example, sterile saline and phosphate-buffered salineat physiological pH may be used. Preservatives, stabilizers, dyes andthe like may be provided in the pharmaceutical composition. For example,sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid may beadded as preservatives. Id. at 1449. In addition, antioxidants andsuspending agents may be used. Id. The compounds of the presentinvention may be used in either the free base or salt forms, with bothforms being considered as being within the scope of the presentinvention.

The pharmaceutical compositions that contain one or more nucleic acidconstructs of the invention, or the proteins corresponding to theproducts encoded by such nucleic acid constructs, may be in any formwhich allows for the composition to be administered to a patient. Forexample, the composition may be in the form of a solid, liquid or gas(aerosol). Typical routes of administration include, without limitation,oral, topical, parenteral (e.g., sublingually or buccally), sublingual,rectal, vaginal, and intranasal. The term parenteral as used hereinincludes subcutaneous injections, intravenous, intramuscular,intrasternal, intracavernous, intrathecal, intrameatal, intraurethralinjection or infusion techniques. The pharmaceutical composition isformulated so as to allow the active ingredients contained therein to bebioavailable upon administration of the composition to a patient.Compositions that will be administered to a patient take the form of oneor more dosage units, where for example, a tablet may be a single dosageunit, and a container of one or more compounds of the invention inaerosol form may hold a plurality of dosage units.

For oral administration, an excipient and/or binder may be present.Examples are sucrose, kaolin, glycerin, starch dextrins, sodiumalginate, carboxymethylcellulose and ethyl cellulose. Coloring and/orflavoring agents may be present. A coating shell may be employed.

The composition may be in the form of a liquid, e.g., an elixir, syrup,solution, emulsion or suspension. The liquid may be for oraladministration or for delivery by injection, as two examples. Whenintended for oral administration, preferred compositions contain, inaddition to one or more binding domain-immunoglobulin fusion constructor expressed product, one or more of a sweetening agent, preservatives,dye/colorant and flavor enhancer. In a composition intended to beadministered by injection, one or more of a surfactant, preservative,wetting agent, dispersing agent, suspending agent, buffer, stabilizerand isotonic agent may be included.

A liquid pharmaceutical composition as used herein, whether in the formof a solution, suspension or other like form, may include one or more ofthe following adjuvants: sterile diluents such as water for injection,saline solution, preferably physiological saline, Ringer's solution,isotonic sodium chloride, fixed oils such as synthetic mono ordigylcerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. The parenteral preparation can be enclosedin ampoules, disposable syringes or multiple dose vials made of glass orplastic. Physiological saline is a preferred adjuvant. An injectablepharmaceutical composition is preferably sterile.

It may also be desirable to include other components in the preparation,such as delivery vehicles including, but not limited to, aluminum salts,water-in-oil emulsions, biodegradable oil vehicles, oil-in-wateremulsions, biodegradable microcapsules, and liposomes. Examples ofimmunostimulatory substances (adjuvants) for use in such vehiclesinclude N-acetylmuramyl-L-alanine-D-isoglutamine (MDP),lipopolysaccharides (LPS), glucan, IL-12, GM-CSF, gamma interferon andIL-15.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of this invention,the type of carrier will vary depending on the mode of administrationand whether a sustained release is desired. For parenteraladministration, such as subcutaneous injection, the carrier preferablycomprises water, saline, alcohol, a fat, a wax or a buffer. For oraladministration, any of the above carriers or a solid carrier, such asmannitol, lactose, starch, magnesium stearate, sodium saccharine,talcum, cellulose, glucose, sucrose, and magnesium carbonate, may beemployed. Biodegradable microspheres (e.g., polylactic galactide) mayalso be employed as carriers for the pharmaceutical compositions of thisinvention. Suitable biodegradable microspheres are disclosed, forexample, in U.S. Pat. Nos. 4,897,268 and 5,075,109. In this regard, itis preferable that the microsphere be larger than approximately 25microns.

Pharmaceutical compositions may also contain diluents such as buffers,antioxidants such as ascorbic acid, low molecular weight (less thanabout 10 residues) polypeptides, proteins, amino acids, carbohydrates(e.g., glucose, sucrose or dextrins), chelating agents (e.g., EDTA),glutathione and other stabilizers and excipients. Neutral bufferedsaline or saline mixed with nonspecific serum albumin are exemplaryappropriate diluents. Preferably, product is formulated as alyophilizate using appropriate excipient solutions (e.g., sucrose) asdiluents.

The pharmaceutical compositions according to the invention also includestabilized proteins and stable liquid pharmaceutical formulations inaccordance with technology known in the art, including the technologydisclosed in Published US Patent Application No. 2006/0008415 A1,incorporated herein by reference. Such technologies includederivatization of a protein, wherein the protein comprises a thiol groupcoupled to N-acetyl-L-cysteine, N-ethyl-maleimide, or cysteine.

As described above, the subject invention includes compositions capableof delivering nucleic acid molecules encoding multivalent bindingproteins with effector function. Such compositions include recombinantviral vectors, e.g., retroviruses (see WO 90/07936, WO 91/02805, WO93/25234, WO 93/25698, and WO 94/03622), adenovirus (see Berkner, 1988Biotechniques 6:616-627; Li et al., 1993 Hum. Gene Ther. 4:403-409;Vincent et al., Nat. Genet. 5:130-134; and Kolls et al., 1994 Proc.Natl. Acad. Sci. USA 91:215-219), pox virus (see U.S. Pat. No.4,769,330; U.S. Pat. No. 5,017,487; and WO 89/01973)), recombinantexpression construct nucleic acid molecules complexed to a polycationicmolecule (see WO 93/03709), and nucleic acids associated with liposomes(see Wang et al., 1987 Proc. Natl. Acad. Sci. USA 84:7851). In certainembodiments, the DNA may be linked to killed or inactivated adenovirus(see Curiel et al., 1992 Hum. Gene Ther. 3:147-154; Cotton et al., 1992Proc. Natl. Acad. Sci. USA 89:6094). Other suitable compositions includeDNA-ligand (see Wu et al., 1989 J. Biol. Chem. 264:16985-16987) andlipid-DNA combinations (see Felgner et al., 1989 Proc. Natl. Acad. Sci.USA 84:7413-7417).

In addition to direct in vivo procedures, ex vivo procedures may be usedin which cells are removed from a host (e.g., a subject, such as a humanpatient), modified, and placed into the same or another host animal. Itwill be evident that one can utilize any of the compositions noted abovefor introduction of constructs of the invention, either theproteins/polypeptides or the nucleic acids encoding them into tissuecells in an ex vivo context. Protocols for viral, physical and chemicalmethods of uptake are well known in the art.

Generation of Antibodies

Polyclonal antibodies directed toward an antigen polypeptide generallyare produced in animals (e.g., rabbits, hamsters, goats, sheep, horses,pigs, rats, gerbils, guinea pigs, mice, or any other suitable mammal, aswell as other non-mammal species) by means of multiple subcutaneous orintraperitoneal injections of antigen polypeptide or a fragment thereofand an adjuvant. Adjuvants include, but are not limited to, complete orincomplete Freund's adjuvant, mineral gels such as aluminum hydroxide,and surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, and dinitrophenol. BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are also potentially usefuladjuvants. It may be useful to conjugate an antigen polypeptide to acarrier protein that is immunogenic in the species to be immunized;typical carriers include keyhole limpet hemocyanin, serum albumin,bovine thyroglobulin, or soybean trypsin inhibitor. Also, aggregatingagents such as alum are used to enhance the immune response. Afterimmunization, the animals are bled and the serum is assayed foranti-antigen polypeptide antibody titer using conventional techniques.Polyclonal antibodies may be utilized in the sera from which they weredetected, or may be purified from the sera using, e.g., antigen affinitychromatography.

Monoclonal antibodies directed toward antigen polypeptides are producedusing any method which provides for the production of antibody moleculesby continuous cell lines in culture. For example, monoclonal antibodiesmay be made by the hybridoma method as described in Kohler et al.,Nature 256:495 [1975]; the human B-cell hybridoma technique (Kosbor etal., Immunol Today 4:72, 1983; Cote et al., Proc Natl Acad Sci 80:2026-2030, 1983) and the EBV-hybridoma technique (Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, New YorkN.Y., pp 77-96, (1985).

When the hybridoma technique is employed, myeloma cell lines may beused. Cell lines suited for use in hybridoma-producing fusion procedurespreferably do not produce endogenous antibody, have high fusionefficiency, and exhibit enzyme deficiencies that render them incapableof growing in certain selective media which support the growth of onlythe desired fused cells (hybridomas). For example, where the immunizedanimal is a mouse, one may use P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 4 1,Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; forrats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266,GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection withcell fusions.

In an alternative embodiment, human antibodies can be produced fromphage-display libraries (Hoogenboom et al., J. Mol. Biol. 227: 381[1991]; Marks et al., J. Mol. Biol. 222: 581, see also U.S. Pat. No.5,885,793)). These processes mimic immune selection through the displayof antibody repertoires on the surface of filamentous bacteriophage, andsubsequent selection of phage by their binding to an antigen of choice.One such technique is described in PCT Application No. PCT/US98/17364,filed in the name of Adams et al., which describes the isolation of highaffinity and functional agonistic antibodies for MPL- and msk-receptorsusing such an approach. In this approach, a complete repertoire of humanantibody genes can be created by cloning naturally rearranged human Vgenes from peripheral blood lymphocytes as previously described(Mullinax, et al., Proc. Natl. Acad. Sci. (USA) 87: 8095-8099 [1990]).

Alternatively, an entirely synthetic human heavy chain repertoire can becreated from unrearranged V gene segments by assembling each human VHsegment with D segments of random nucleotides together with a human Jsegment (Hoogenboom, et al., J. Mol. Biol. 227:381-388 [1992]).Likewise, a light chain repertoire can be constructed by combining eachhuman V segment with a J segment (Griffiths, et al, EMBO J. 13:3245-3260[1994]). Nucleotides encoding the complete antibody (i.e., both heavyand light chains) are linked as a single-chain Fv fragment and thispolynucleotide is ligated to a nucleotide encoding a filamentous phageminor coat protein. When this fusion protein is expressed on the surfaceof the phage, a polynucleotide encoding a specific antibody can beidentified by selection using an immobilized antigen.

Beyond the classic methods of generating polyclonal and monoclonalantibodies, any method for generating any known antibody form iscontemplated. In addition to polyclonals and monoclonals, antibody formsinclude chimerized antibodies, humanized antibodies, CDR-graftedantibodies, and antibody fragments and variants.

Variants and Derivatives of Specific Binding Agents

In one example, insertion variants are provided wherein one or moreamino acid residues supplement a specific binding agent amino acidsequence. Insertions may be located at either or both termini of theprotein, or may be positioned within internal regions of the specificbinding agent amino acid sequence. Variant products of the inventionalso include mature specific binding agent products, i.e., specificbinding agent products wherein leader or signal sequences are removed,and the resulting protein having additional amino terminal residues. Theadditional amino terminal residues may be derived from another protein,or may include one or more residues that are not identifiable as beingderived from a specific protein. Polypeptides with an additionalmethionine residue at position −1 (e.g., Met-1-multivalent bindingpeptides with effector function) are contemplated, as are polypeptidesof the invention with additional methionine and lysine residues atpositions −2 and −1 (Met-2-Lys-1-multivalent binding proteins witheffector function). Variants of the polypeptides of the invention havingadditional Met, Met-Lys, or Lys residues (or one or more basic residuesin general) are particularly useful for enhanced recombinant proteinproduction in bacterial host cells.

The invention also embraces specific polypeptides of the inventionhaving additional amino acid residues which arise from use of specificexpression systems. For example, use of commercially available vectorsthat express a desired polypeptide as part of aglutathione-S-transferase (GST) fusion product provides the desiredpolypeptide having an additional glycine residue at position −1 aftercleavage of the GST component from the desired polypeptide. Variantswhich result from expression in other vector systems are alsocontemplated, including those wherein histidine tags are incorporatedinto the amino acid sequence, generally at the carboxy and/or aminoterminus of the sequence.

In another aspect, the invention provides deletion variants wherein oneor more amino acid residues in a polypeptide of the invention areremoved. Deletions can be effected at one or both termini of thepolypeptide, or from removal of one or more residues within the aminoacid sequence. Deletion variants necessarily include all fragments of apolypeptide according to the invention.

Antibody fragments refer to polypeptides having a sequence correspondingto at least part of an immunoglobulin variable region sequence.Fragments may be generated, for example, by enzymatic or chemicalcleavage of polypeptides corresponding to full-length antibodies. Otherbinding fragments include those generated by synthetic techniques or byrecombinant DNA techniques, such as the expression of recombinantplasmids containing nucleic acid sequences encoding partial antibodyvariable regions. Preferred polypeptide fragments display immunologicalproperties unique to, or specific for, a target as described herein.Fragments of the invention having the desired immunological propertiescan be prepared by any of the methods well known and routinely practicedin the art.

In still another aspect, the invention provides substitution variants ofmultivalent binding polypeptides having effector function. Substitutionvariants include those polypeptides wherein one or more amino acidresidues in an amino acid sequence are removed and replaced withalternative residues. In some embodiments, the substitutions areconservative in nature; however, the invention embraces substitutionsthat ore also non-conservative. Amino acids can be classified accordingto physical properties and contribution to secondary and tertiaryprotein structure. A conservative substitution is recognized in the artas a substitution of one amino acid for another amino acid that hassimilar properties. Exemplary conservative substitutions are set out inTable A (see WO 97/09433, page 10, published Mar. 13, 1997(PCT/GB96/02197, filed Sep. 6, 1996), immediately below.

TABLE A Conservative Substitutions I SIDE CHAIN CHARACTERISTIC AMINOACID Aliphatic Non-polar G A P I L V Polar - uncharged S T M N Q Polar -charged D E K R Aromatic H F W Y Other N Q D E

Alternatively, conservative amino acids can be grouped as described inLehninger, [Biochemistry, Second Edition; Worth Publishers, Inc. NY:N.Y.(1975), pp. 71-77] as set out in Table B, immediately below.

TABLE B Conservative Substitutions II SIDE CHAIN CHARACTERISTIC AMINOACID Non-polar A. Aliphatic: A L I V P (hydrophobic) B. Aromatic F W C.Sulfur-containing M D. Borderline G Uncharged-polar A. Hydroxyl S T Y B.Amides N Q C. Sulfhydryl C D. Borderline G Positively Charged K R H(Basic) Negatively D E Charged (Acidic)

Conservative Substitutions II

SIDE CHAIN CHARACTERISTIC AMINO ACID Non-polar A. Aliphatic: A L I V P(hydrophobic) B. Aromatic: F W C. Sulfur-containing: M D. Borderline: GUncharged-polar A. Hydroxyl: S T Y B. Amides: N Q C. Sulfhydryl: C D.Borderline: G Positively Charged K R H (Basic) Negatively Charged D E(Acidic)

The invention also provides derivatives of specific binding agentpolypeptides. Derivatives include specific binding agent polypeptidesbearing modifications other than insertion, deletion, or substitution ofamino acid residues. Preferably, the modifications are covalent innature, and include for example, chemical bonding with polymers, lipids,other organic, and inorganic moieties. Derivatives of the invention maybe prepared to increase circulating half-life of a specific bindingagent polypeptide, or may be designed to improve targeting capacity forthe polypeptide to desired cells, tissues, or organs.

The invention further embraces multivalent binding proteins witheffector function that are covalently modified or derivatized to includeone or more water-soluble polymer attachments such as polyethyleneglycol, polyoxyethylene glycol, or polypropylene glycol, as describedU.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 and4,179,337. Still other useful polymers known in the art includemonomethoxy-polyethylene glycol, dextran, cellulose, and othercarbohydrate-based polymers, poly-(N-vinyl pyrrolidone)-polyethyleneglycol, propylene glycol homopolymers, a polypropylene oxide/ethyleneoxide co-polymer, polyoxyethylated polyols (e.g., glycerol) andpolyvinyl alcohol, as well as mixtures of these polymers. Particularlypreferred are polyethylene glycol (PEG)-derivatized proteins.Water-soluble polymers may be bonded at specific positions, for exampleat the amino terminus of the proteins and polypeptides according to theinvention, or randomly attached to one or more side chains of thepolypeptide. The use of PEG for improving therapeutic capacities isdescribed in U.S. Pat. No. 6,133,426 to Gonzales, et al.

Target Sites for Immunoglobulin Mutagenesis

Certain strategies are available to manipulate inherent properties of anantigen-specific immunoglobulin (e.g., an antibody) that are notavailable to non-immunoglobulin-based binding molecules. A good exampleof the strategies favoring, e.g., antibody-based molecules, over thesealternatives is the in vivo modulation of the affinity of an antibodyfor its target through affinity maturation, which takes advantage of thesomatic hypermutation of immunoglobulin genes to yield antibodies ofincreasing affinity as an immune response progresses. Additionally,recombinant technologies have been developed to alter the structure ofimmunoglobulins and immunoglobulin regions and domains. Thus,polypeptides derived from antibodies may be produced that exhibitaltered affinity for a given antigen, and a number of purificationprotocols and monitoring screens are known in the art for identifyingand purifying or isolating these polypeptides. Using these knowntechniques, polypeptides comprising antibody-derived binding domains canbe obtained that exhibit decreased or increased affinity for an antigen.Strategies for generating the polypeptide variants exhibiting alteredaffinity include the use of site-specific or random mutagenesis of theDNA encoding the antibody to change the amino acids present in theprotein, followed by a screening step designed to recover antibodyvariants that exhibit the desired change, e.g., increased or decreasedaffinity relative to the unmodified parent or referent antibody.

The amino acid residues most commonly targeted in mutagenic strategiesto alter affinity are those in the complementarity-determining region(CDR) or hyper-variable region of the light and the heavy chain variableregions of an antibody. These regions contain the residues thatphysicochemically interact with an antigen, as well as other amino acidsthat affect the spatial arrangement of these residues. However, aminoacids in the framework regions of the variable domains outside the CDRregions have also been shown to make substantial contributions to theantigen-binding properties of an antibody, and can be targeted tomanipulate such properties. See Hudson, P. J. Curr. Opin. Biotech., 9:395-402 (1999) and references therein.

Smaller and more effectively screened libraries of antibody variants canbe produced by restricting random or site-directed mutagenesis to sitesin the CDRs that correspond to areas prone to “hyper-mutation” duringthe somatic affinity maturation process. See Chowdhury, et al., NatureBiotech., 17: 568-572 (1999) and references therein. The types of DNAelements known to define hyper-mutation sites in this manner includedirect and inverted repeats, certain consensus sequences, secondarystructures, and palindromes. The consensus DNA sequences include thetetrabase sequence Purine-G-Pyrimidine-A/T (i.e., A or G-G-C or T-A orT) and the serine codon AGY (wherein Y can be C or T).

Thus, another aspect of the invention is a set of mutagenic strategiesfor modifying the affinity of an antibody for its target. Thesestrategies include mutagenesis of the entire variable region of a heavyand/or light chain, mutagenesis of the CDR regions only, mutagenesis ofthe consensus hypermutation sites within the CDRs, mutagenesis offramework regions, or any combination of these approaches (“mutagenesis”in this context could be random or site-directed). Definitivedelineation of the CDR regions and identification of residues comprisingthe binding site of an antibody can be accomplished though solving thestructure of the antibody in question, and the antibody:ligand complex,through techniques known to those skilled in the art, such as X-raycrystallography. Various methods based on analysis and characterizationof such antibody crystal structures are known to those of skill in theart and can be employed to approximate the CDR regions. Examples of suchcommonly used methods include the Kabat, Chothia, AbM and contactdefinitions.

The Kabat definition is based on sequence variability and is the mostcommonly used definition to predict CDR regions. Johnson, et al.,Nucleic Acids Research, 28: 214-8 (2000). The Chothia definition isbased on the location of the structural loop regions. (Chothia et al.,J. Mol. Biol., 196: 901-17 [1986]; Chothia et al., Nature, 342: 877-83[1989].) The AbM definition is a compromise between the Kabat andChothia definitions. AbM is an integral suite of programs for antibodystructure modeling produced by the Oxford Molecular Group (Martin, etal., Proc. Natl. Acad. Sci (USA) 86:9268-9272 [1989]; Rees, et al.,ABMTM, a computer program for modeling variable regions of antibodies,Oxford, UK; Oxford Molecular, Ltd.). The AbM suite models the tertiarystructure of an antibody from primary sequence using a combination ofknowledge databases and ab initio methods An additional definition,known as the contact definition, has been recently introduced. SeeMacCallum et al., J. Mol. Biol., 5:732-45 (1996). This definition isbased on an analysis of the available complex crystal structures.

By convention, the CDR domains in the heavy chain are typically referredto as H1, H2 and H3, and are numbered sequentially in order moving fromthe amino terminus to the carboxy terminus. The CDR regions in the lightchain are typically referred to as L1, L2 and L3, and are numberedsequentially in order moving from the amino terminus to the carboxyterminus.

The CDR-H1 is approximately 10 to 12 residues in length and typicallystarts 4 residues after a Cys according to the Chothia and AbMdefinitions, or typically 5 residues later according to the Kabatdefinition. The H1 is typically followed by a Trp, typically Trp-Val,but also Trp-Ile, or Trp-Ala. The length of H1 is approximately 10 to 12residues according to the AbM definition, while the Chothia definitionexcludes the last 4 residues.

The CDR-H2 typically starts 15 residues after the end of H1 according tothe Kabat and AbM definitions. The residues preceding H2 are typicallyLeu-Glu-Trp-Ile-Gly but there are a number of variations. H2 istypically followed by the amino acid sequenceLys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala. According to the Kabatdefinition, the length of H2 is approximately 16 to 19 residues, wherethe AbM definition predicts the length to be typically 9 to 12 residues.

The CDR-H3 typically starts 33 residues after the end of H2 and istypically preceded by the amino acid sequence Cys-Ala-Arg. H3 istypically followed by the amino acid Gly. The length of H3 ranges from 3to 25 residues

The CDR-L 1 typically starts at approximately residue 24 and willtypically follow a Cys. The residue after the CDR-L1 is always Trp andwill typically begin one of the following sequences: Trp-Tyr-Gln,Trp-Leu-Gln, Trp-Phe-Gln, or Trp-Tyr-Leu. The length of CDR-L1 isapproximately 10 to 17 residues.

The CDR-L2 starts approximately 16 residues after the end of L1. It willgenerally follow residues Ile-Tyr, Val-Tyr, Ile-Lys or Ile-Phe. Thelength of CDR-L2 is approximately 7 residues.

The CDR-L3 typically starts 33 residues after the end of L2 andtypically follows a Cys. L3 is typically followed by the amino acidsequence Phe-Gly-XXX-Gly. The length of L3 is approximately 7 to 11residues.

Various methods for modifying antibodies have been described in the art,including, e.g., methods of producing humanized antibodies wherein thesequence of the humanized immunoglobulin heavy chain variable regionframework is 65% to 95% identical to the sequence of the donorimmunoglobulin heavy chain variable region framework. Each humanizedimmunoglobulin chain will usually comprise, in addition to the CDRs,amino acids from the donor immunoglobulin framework that are, e.g.,capable of interacting with the CDRs to effect binding affinity, such asone or more amino acids that are immediately adjacent to a CDR in thedonor immunoglobulin or those within about 3 angstroms, as predicted bymolecular modeling. The heavy and light chains may each be designed byusing any one or all of various position criteria. When combined into anintact antibody, humanized immunoglobulins are substantiallynon-immunogenic in humans and retain substantially the same affinity asthe donor immunoglobulin to the antigen, such as a protein or othercompound containing an epitope.

In one example, methods for the production of antibodies, and antibodyfragments, are described that have binding specificity similar to aparent antibody, but which have increased human characteristics.Humanized antibodies are obtained by chain shuffling using, for example,phage display technology and a polypeptide comprising the heavy or lightchain variable region of a non-human antibody specific for an antigen ofinterest, which is then combined with a repertoire of humancomplementary (light or heavy) chain variable regions. Hybrid pairingswhich are specific for the antigen of interest are identified and humanchains from the selected pairings are combined with a repertoire ofhuman complementary variable domains (heavy or light). In anotherembodiment, a component of a CDR from a non-human antibody is combinedwith a repertoire of component parts of CDRs from human antibodies. Fromthe resulting library of antibody polypeptide dimers, hybrids areselected and may used in a second humanizing shuffling step;alternatively, this second step is eliminated if the hybrid is alreadyof sufficient human character to be of therapeutic value. Methods ofmodification to increase human character are known in the art.

Another example is a method for making humanized antibodies bysubstituting a CDR amino acid sequence for the corresponding human CDRamino acid sequence and/or substituting a FR amino acid sequence for thecorresponding human FR amino acid sequences.

Yet another example provides methods for identifying the amino acidresidues of an antibody variable domain that may be modified withoutdiminishing the native affinity of the antigen binding domain whilereducing its immunogenicity with respect to a heterologous species andmethods for preparing these modified antibody variable regions as usefulfor administration to heterologous species.

Modification of an immunoglobulin such as an antibody by any of themethods known in the art is designed to achieve increased or decreasedbinding affinity for an antigen and/or to reduce immunogenicity of theantibody in the recipient and/or to modulate effector activity levels.In one approach, humanized antibodies can be modified to eliminateglycosylation sites in order to increase affinity of the antibody forits cognate antigen (Co, et al., Mol. Immunol. 30:1361-1367 [1993]).Techniques such as “reshaping,” hyperchimerization,” and“veneering/resurfacing” have produced humanized antibodies with greatertherapeutic potential. Vaswami, et al., Annals of Allergy, Asthma, &Immunol 81:105 (1998); Roguska, et al., Prot. Engineer. 9:895-904(1996)]. See also U.S. Pat. No. 6,072,035, which describes methods forreshaping antibodies. While these techniques diminish antibodyimmunogenicity by reducing the number of foreign residues, they do notprevent anti-idiotypic and anti-allotypic responses following repeatedadministration of the antibodies. Alternatives to these methods forreducing immunogenicity are described in Gilliland et al., J. Immunol.62(6):3663-71 (1999).

In many instances, humanizing antibodies results in a loss of antigenbinding capacity. It is therefore preferable to “back mutate” thehumanized antibody to include one or more of the amino acid residuesfound in the original (most often rodent) antibody in an attempt torestore binding affinity of the antibody. See, for example, Saldanha etal., Mol. Immunol. 36:709-19 (1999).

Glycosylation of immunoglobulins has been shown to affect effectorfunctions, structural stability, and the rate of secretion fromantibody-producing cells (see Leatherbarrow et al., Mol. Immunol. 22:407(1985), incorporated herein by reference). The carbohydrate groupsresponsible for these properties are generally attached to the constantregions of antibodies. For example, glycosylation of IgG at Asn 297 inthe C_(H2) domain facilitates full capacity of the IgG to activatecomplement-dependent cytolysis (Tao et al., J. Immunol. 143:2595(1989)). Glycosylation of IgM at Asn 402 in the C_(H3) domain, forexample, facilitates proper assembly and cytolytic activity of theantibody (Muraoka et al., J. Immunol. 142:695 (1989)). Removal ofglycosylation sites at positions 162 and 419 in the C_(H1) and C_(H3)domains of an IgA antibody led to intracellular degradation and at least90% inhibition of secretion (Taylor et al., Wall, Mol. Cell. Biol.8:4197 (1988)). Accordingly, the molecules of the invention includemutationally altered immunoglobulins exhibiting altered glycosylationpatterns by mutation of specific residues in, e.g., a constantsub-region to alter effector function. See Co et al., Mol. Immunol.30:1361-1367 (1993), Jacquemon et al., J. Thromb. Haemost. 4:1047-1055(2006), Schuster et al., Cancer Res. 65:7934-7941 (2005), and Warnock etal., Biotechnol Bioeng. 92:831-842 (2005), each incorporated herein byreference.

The invention also includes multivalent binding molecules having atleast one binding domain that is at least 80%, preferably 90% or 95% or99% identical in sequence to a known immunoglobulin variable regionsequence and which has at least one residue that differs from suchimmunoglobulin variable region, wherein the changed residue adds aglycosylation site, changes the location of one or more glycosylationsite(s), or preferably removes a glycosylation site relative to theimmunoglobulin variable region. In some embodiments, the change removesan N-linked glycosylation site in a an immunoglobulin variable regionframework, or removes an N-linked glycosylation site that occurs in theimmunoglobulin heavy chain variable region framework in the regionspanning about amino acid residue 65 to about amino acid residue 85,using the numbering convention of Co et al., J. Immunol. 148: 1149,(1992).

Any method known in the art is contemplated for producing themultivalent binding molecules exhibiting altered glycosylation patternsrelative to an immunoglobulin referent sequence. For example, any of avariety of genetic techniques may be employed to alter one or moreparticular residues. Alternatively, the host cells used for productionmay be engineered to produce the altered glycosylation pattern. Onemethod known in the art, for example, provides altered glycosylation inthe form of bisected, non-fucosylated variants that increase ADCC. Thevariants result from expression in a host cell containing anoligosaccharide-modifying enzyme. Alternatively, the Potelligenttechnology of BioWa/Kyowa Hakko is contemplated to reduce the fucosecontent of glycosylated molecules according to the invention. In oneknown method, a CHO host cell for recombinant immunoglobulin productionis provided that modifies the glycosylation pattern of theimmunoglobulin F_(C) region, through production of GDP-fucose. Thistechnology is available to modify the glycosylation pattern of aconstant sub-region of a multivalent binding molecule according to theinvention.

In addition to modifying the binding properties of binding domains, suchas the binding domains of immunoglobulins, and in addition to suchmodifications as humanization, the invention comprehends the modulationof effector function by changing or mutating residues contributing toeffector function, such as the effector function of a constantsub-region. These modifications can be effected using any techniqueknown in the art, such as the approach disclosed in Presta et al.,Biochem. Soc. Trans. 30:487-490 (2001), incorporated herein byreference. Exemplary approaches would include the use of the protocoldisclosed in Presta et al. to modify specific residues known to affectbinding in one or more constant sub-regions corresponding to FCγRI,FCγRII, FCγRIII, FCαR, and FCεR.

In another approach, the Xencor XmAb technology is available to engineerconstant sub-regions corresponding to F_(C) domains to enhance cellkilling effector function. See Lazar et al., Proc. Natl. Acad. Sci.(USA) 103(11):4005-4010 (2006), incorporated herein by reference. Usingthis approach, for example, one can generate constant sub-regionsoptimized for F_(C)γR specificity and binding, thereby enhancing cellkilling effector function.

Production of Multivalent Binding Proteins with Effector Function

A variety of expression vector/host systems may be utilized to containand express the multivalent binding protein (with effector function) ofthe invention. These systems include but are not limited tomicroorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, cosmid, or other expression vectors; yeasttransformed with yeast expression or shuttle vectors; insect cellsystems infected with virus expression vectors (e.g., baculovirus);plant cell systems transfected with virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with bacterial expression vectors (e.g., Ti or pBR322plasmid); or animal cell systems. Mammalian cells that are useful inrecombinant multivalent binding protein productions include, but are notlimited to, VERO cells, HeLa cells, Chinese hamster ovary (CHO) celllines, COS cells (such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK,A549, PC12, K562 and HEK293 cells. Exemplary protocols for therecombinant expression of the multivalent binding protein are describedherein below.

An expression vector can comprise a transcriptional unit comprising anassembly of (1) a genetic element or elements having a regulatory rolein gene expression, for example, a promoter, enhancer, orfactor-specific binding site, (2) a structural or sequence that encodesthe binding agent which is transcribed into mRNA and translated intoprotein, and (3) appropriate transcription initiation and terminationsequences. Structural units intended for use in yeast or eukaryoticexpression systems preferably include a leader sequence enablingextracellular secretion of translated protein by a host cell.Alternatively, where recombinant multivalent binding protein isexpressed without a leader or transport sequence, it may include anamino terminal methionine residue. This residue may or may not besubsequently cleaved from the expressed recombinant protein to provide afinal multivalent binding protein.

For example, the multivalent binding proteins may be recombinantlyexpressed in yeast using a commercially available expression system,e.g., the Pichia Expression System (Invitrogen, San Diego, Calif.),following the manufacturer's instructions. This system also relies onthe pre-pro-alpha sequence to direct secretion, but transcription of theinsert is driven by the alcohol oxidase (AOX1) promoter upon inductionby methanol. The secreted multivalent binding peptide may be purifiedfrom the yeast growth medium by, e.g., the methods used to purify thepeptide from bacterial and mammalian cell supernatants.

Alternatively, the cDNA encoding the multivalent binding peptide may becloned into the baculovirus expression vector pVL1393 (PharMingen, SanDiego, Calif.). This vector can be used according to the manufacturer'sdirections (PharMingen) to infect Spodoptera frugiperda cells in SF9protein-free medium and to produce recombinant protein. The multivalentbinding protein can be purified and concentrated from the medium using aheparin-Sepharose column (Pharmacia, Piscataway, N.J.). Insect systemsfor protein expression, such as the SF9 system, are well known to thoseof skill in the art. In one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) can be used as a vector to express foreigngenes in the Spodoptera frugiperda cells or in Trichoplusia larvae. Themultivalent binding peptide coding sequence can be cloned into anonessential region of the virus, such as the polyhedrin gene, andplaced under control of the polyhedrin promoter. Successful insertion ofthe multivalent binding peptide will render the polyhedrin gene inactiveand produce recombinant virus lacking coat protein. The recombinantviruses can be used to infect S. frugiperda cells or Trichoplusia larvaein which peptide is expressed (Smith et al., J Virol 46: 584, 1983;Engelhard et al., Proc Nat Acad Sci (USA) 91: 3224-7, 1994).

In another example, the DNA sequence encoding the multivalent bindingpeptide can be amplified by PCR and cloned into an appropriate vector,for example, pGEX-3X (Pharmacia, Piscataway, N.J.). The pGEX vector isdesigned to produce a fusion protein comprisingglutathione-S-transferase (GST), encoded by the vector, and amultivalent binding protein encoded by a DNA fragment inserted into thecloning site of the vector. The primers for the PCR can be generated toinclude for example, an appropriate cleavage site. Where the multivalentbinding protein fusion moiety is used solely to facilitate expression oris otherwise not desirable as an attachment to the peptide of interest,the recombinant multivalent binding protein fusion may then be cleavedfrom the GST portion of the fusion protein. The pGEX-3X/multivalentbinding peptide construct is transformed into E. coli XL-1 Blue cells(Stratagene, La Jolla Calif.), and individual transformants isolated andgrown. Plasmid DNA from individual transformants is purified and may bepartially sequenced using an automated sequencer to confirm the presenceof the desired multivalent binding protein-encoding nucleic acid insertin the proper orientation.

The fused multivalent binding protein, which may be produced as aninsoluble inclusion body in the bacteria, can be purified as follows.Host cells can be harvested by centrifugation; washed in 0.15 M NaCl, 10mM Tris, pH 8, 1 mM EDTA; and treated with 0.1 mg/ml lysozyme (SigmaChemical Co.) for 15 minutes at room temperature. The lysate can becleared by sonication, and cell debris can be pelleted by centrifugationfor 10 minutes at 12,000×g. The multivalent binding proteinfusion-containing pellet can be resuspended in 50 mM Tris, pH 8, and 10mM EDTA, layered over 50% glycerol, and centrifuged for 30 minutes at6000 g. The pellet can be resuspended in standard phosphate bufferedsaline solution (PBS) free of Mg⁺⁺ and Ca⁺⁺. The multivalent bindingprotein fusion can be further purified by fractionating the resuspendedpellet in a denaturing SDS polyacrylamide gel (Sambrook et al.). The gelis soaked in 0.4 M KCl to visualize the protein, which is excised andelectroeluted in gel-running buffer lacking SDS. If the GST/multivalentbinding peptide fusion protein is produced in bacteria as a solubleprotein, it can be purified using the GST Purification Module (PharmaciaBiotech).

The multivalent binding protein fusion is preferably subjected todigestion to cleave the GST from the multivalent binding peptide of theinvention. The digestion reaction (20-40 μg fusion protein, 20-30 unitshuman thrombin (4000 U/mg (Sigma) in 0.5 ml PBS) can be incubated 16-48hours at room temperature and loaded on a denaturing SDS-PAGE gel tofractionate the reaction products. The gel can be soaked in 0.4 M KCl tovisualize the protein bands. The identity of the protein bandcorresponding to the expected molecular weight of the multivalentbinding peptide can be confirmed by amino acid sequence analysis usingan automated sequencer (Applied Biosystems Model 473A, Foster City,Calif.). Alternatively, the identity can be confirmed by performing HPLCand/or mass spectrometry of the peptides.

Alternatively, a DNA sequence encoding the multivalent binding peptidecan be cloned into a plasmid containing a desired promoter and,optionally, a leader sequence (see, e.g., Better et al., Science,240:1041-43, 1988). The sequence of this construct can be confirmed byautomated sequencing. The plasmid can then be transformed into asuitable E. coli strain, such as strain MC1061, using standardprocedures employing CaCl₂ incubation and heat shock treatment of thebacteria (Sambrook et al.). The transformed bacteria can be grown in LBmedium supplemented with carbenicillin or another suitable form ofselection as would be known in the art, and production of the expressedprotein can be induced by growth in a suitable medium. If present, theleader sequence can effect secretion of the multivalent binding peptideand be cleaved during secretion. The secreted recombinant protein can bepurified from the bacterial culture medium by the methods describedherein below.

Mammalian host systems for the expression of the recombinant protein arewell known to those of skill in the art and are preferred systems. Hostcell strains can be chosen for a particular ability to process theexpressed protein or produce certain post-translation modifications thatwill be useful in providing protein activity. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation. Different hostcells such as CHO, HeLa, MDCK, 293, WI38, and the like, have specificcellular machinery and characteristic mechanisms for suchpost-translational activities and can be chosen to ensure the correctmodification and processing of the foreign protein.

It is preferable that the transformed cells be used for long-term,high-yield protein production and, as such, stable expression isdesirable. Once such cells are transformed with vectors that preferablycontain at least one selectable marker along with the desired expressioncassette, the cells are grown for 1-2 days in an enriched medium beforebeing switched to selective medium. The selectable marker is designed toconfer resistance to selection and its presence allows growth andrecovery of cells that successfully express the foreign protein.Resistant clumps of stably transformed cells can be proliferated usingtissue culture techniques appropriate to the cell.

A number of selection systems can be used to recover the cells that havebeen transformed for recombinant protein production. Such selectionsystems include, but are not limited to, HSV thymidine kinase,hypoxanthine-guanine phosphoribosyltransferase and adeninephosphoribosyltransferase genes, in tk−, hgprt− or aprt− cells,respectively. Also, anti-metabolite resistance can be used as the basisof selection for dhfr, which confers resistance to methotrexate; gpt,which confers resistance to mycophenolic acid; neo, which confersresistance to the aminoglycoside G418 and confers resistance tochlorsulfuron; and hygro, which confers resistance to hygromycin.Additional selectable genes that may be useful include trpB, whichallows cells to utilize indole in place of tryptophan, or hisD, whichallows cells to utilize histinol in place of histidine. Markers thatgive a visual indication for identification of transformants includeanthocyanins, β-glucuronidase and its substrate, GUS, and luciferase andits substrate, luciferin.

Purification of Proteins

Protein purification techniques are well known to those of skill in theart. These techniques involve, at one level, the crude fractionation ofthe polypeptide and non-polypeptide fractions. Having separated themultivalent binding polypeptide from at least one other protein, thepolypeptide of interest is purified, but further purification usingchromatographic and electrophoretic techniques to achieve partial orcomplete purification (or purification to homogeneity) is frequentlydesired. Analytical methods particularly suited to the preparation of apure multivalent binding peptide are ion-exchange chromatography,exclusion chromatography; polyacrylamide gel electrophoresis; andisoelectric focusing. Particularly efficient methods of purifyingpeptides are fast protein liquid chromatography and HPLC.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of an encodedmultivalent binding protein or peptide. The term “purified multivalentbinding protein or peptide” as used herein, is intended to refer to acomposition, isolatable from other components, wherein the multivalentbinding protein or peptide is purified to any degree relative to itsnaturally obtainable state. A purified multivalent binding protein orpeptide therefore also refers to a multivalent binding protein orpeptide, free from the environment in which it may naturally occur.

Generally, “purified” will refer to a multivalent binding proteincomposition that has been subjected to fractionation to remove variousother components, and which composition substantially retains itsexpressed biological activity. Where the term “substantially purified”is used, this designation refers to a multivalent binding proteincomposition in which the multivalent binding protein or peptide formsthe major component of the composition, such as constituting about 50%,about 60%, about 70%, about 80%, about 90%, about 95%, about 99% or moreof the protein, by weight, in the composition.

Various methods for quantifying the degree of purification of themultivalent binding protein will be known to those of skill in the artin light of the present disclosure. These include, for example,determining the specific binding activity of an active fraction, orassessing the amount of multivalent binding polypeptides within afraction by SDS/PAGE analysis. A preferred method for assessing thepurity of a multivalent binding protein fraction is to calculate thebinding activity of the fraction, to compare it to the binding activityof the initial extract, and to thus calculate the degree ofpurification, herein assessed by a “-fold purification number.” Theactual units used to represent the amount of binding activity will, ofcourse, be dependent upon the particular assay technique chosen tofollow the purification and whether or not the expressed multivalentbinding protein or peptide exhibits a detectable binding activity.

Various techniques suitable for use in multivalent binding proteinpurification are well known to those of skill in the art. These include,for example, precipitation with ammonium sulfate, PEG, antibodies andthe like, or by heat denaturation, followed by centrifugation;chromatography steps such as ion exchange, gel filtration, reversephase, hydroxylapatite and affinity chromatography; isoelectricfocusing; gel electrophoresis; and combinations of these and othertechniques. As is generally known in the art, it is believed that theorder of conducting the various purification steps may be changed, orthat certain steps may be omitted, and still result in a suitable methodfor the preparation of a substantially purified multivalent bindingprotein.

There is no general requirement that the multivalent binding proteinalways be provided in its most purified state. Indeed, it iscontemplated that less substantially multivalent binding proteins willhave utility in certain embodiments. Partial purification may beaccomplished by using fewer purification steps in combination, or byutilizing different forms of the same general purification scheme. Forexample, it is appreciated that a cation-exchange column chromatographyperformed utilizing an HPLC apparatus will generally result in greaterpurification than the same technique utilizing a low pressurechromatography system. Methods exhibiting a lower degree of relativepurification may have advantages in total recovery of multivalentbinding protein product, or in maintaining binding activity of anexpressed multivalent binding protein.

It is known that the migration of a polypeptide can vary, sometimessignificantly, with different conditions of SDS/PAGE (Capaldi et al.,Biochem. Biophys. Res. Comm., 76:425, 1977). It will therefore beappreciated that under differing electrophoresis conditions, theapparent molecular weights of purified or partially purified multivalentbinding protein expression products may vary.

Effector Cells

Effector cells for inducing, e.g., ADCC, ADCP (antibody-dependentcellular phagocytosis), and the like, against a target cell includehuman leukocytes, macrophages, monocytes, activated neutrophils,activated natural killer (NK) cells, and eosinophils. Effector cellsexpress F_(C)αR (CD89), FcγRI, FcγRII, FcγRIII, and/or F_(C)εR1 andinclude, for example, monocytes and activated neutrophils. Expression ofFcγRI, e.g., has been found to be up-regulated by interferon gamma(IFN-γ). This enhanced expression increases the cytotoxic activity ofmonocytes and neutrophils against target cells. Accordingly, effectorcells may be activated with (IFN-γ) or other cytokines (e.g., TNF-α orβ, colony stimulating factor, IL-2) to increase the presence of FcγRI onthe surface of the cells prior to being contacted with a multivalentprotein of the invention.

The multivalent proteins of the invention provide an antibody effectorfunction, such as antibody-dependent effector cell-mediated cytotoxicity(ADCC), for use against a target cell. Multivalent proteins witheffector function are administered alone, as taught herein, or afterbeing coupled to an effector cell, thereby forming an “activatedeffector cell.” An “activated effector cell” is an effector cell, asdefined herein, linked to a multivalent protein with effector function,also as defined herein, such that the effector cell is effectivelyprovided with a targeting function prior to administration.

Activated effector cells are administered in vivo as a suspension ofcells in a physiologically acceptable solution. The number of cellsadministered is on the order of 10⁸-10⁹, but will vary depending on thetherapeutic purpose. In general, the amount will be sufficient to obtainlocalization of the effector cell at the target cell, and to provide adesired level of effector cell function in that locale, such as cellkilling by ADCC and/or phagocytosis. The term physiologically acceptablesolution, as used herein, is intended to include any carrier solutionwhich stabilizes the targeted effector cells for administration in vivoincluding, for example, saline and aqueous buffer solutions, solvents,antibacterial and antifungal agents, isotonic agents, and the like.

Accordingly, another aspect of the invention provides a method ofinducing a specific antibody effector function, such as ADCC, against acell in a subject, comprising administering to the subject a multivalentprotein (or encoding nucleic acid) or activated effector cell in aphysiologically acceptable medium. Routes of administration can vary andsuitable administration routes will be determined by those of skill inthe art based on a consideration of case-specific variables and routineprocedures, as is known in the art.

Cell-Free Effects

Cell-free effects are also provided by the multivalent molecules of theinvention, e.g., by providing a CDC functionality. The complement systemis a biochemical cascade of the immune system that helps clear foreignmatter such as pathogens from an organism. It is derived from many smallplasma proteins that work together in inducing cytolysis of a targetcell by disrupting the target cell's plasma membrane. The complementsystem consists of more than 35 soluble and cell-bound proteins, 12 ofwhich are directly involved in the complement pathways. The proteins areactive in three biochemical pathways leading to the activation of thecomplement system: the classical complement pathway, the alternatecomplement pathway, and the mannose-binding lectin pathway. Antibodies,in particular the IgG1 class, can also “fix” complement. A detailedunderstanding of these pathways has been achieved in the art and willnot be repeated here, but it is worth noting that complement-dependentcytotoxicity is not dependent on the interaction of a binding moleculewith a cell, e.g., a B cell, of the immune system. Also worth noting isthat the complement system is regulated by complement regulatingproteins. These proteins are present at higher concentrations in theblood plasma than the complement proteins. The complement regulatingproteins are found on the surfaces of self-cells, providing a mechanismto prevent self-cells from being targeted by complement proteins. It isexpected that the complement system plays a role in several diseaseswith an immune component, such as Barraquer-Simons Syndrome, Alzheimer'sdisease, asthma, lupus erythematosus, various forms of arthritis,autoimmune heart disease, and multiple sclerosis. Deficiencies in theterminal pathway predispose an individual to both autoimmune disease andinfections (particularly meningitis).

Diseases, Disorders and Conditions

The invention provides a multivalent binding proteins with effectorfunction, and variant and derivative thereof, that bind to one or morebinding partners and those binding events are useful in the treatment,prevention, or amelioration of a symptom associated with a disease,disorder or pathological condition, preferably one afflicting humans. Inpreferred embodiments of these methods, the multivalent (andmultispecific) binding protein with effector function associates a cellbearing a target, such as a tumor-specific cell-surface marker, with aneffector cell, such as a cell of the immune system exhibiting cytotoxicactivity. In other embodiments, the multispecific, multivalent bindingprotein with effector function specifically binds two differentdisease-, disorder- or condition-specific cell-surface markers to ensurethat the correct target is associated with an effector cell, such as acytotoxic cell of the immune system. Additionally, the multivalentbinding protein with effector function can be used to induce or increaseantigen activity, or to inhibit antigen activity. The multivalentbinding proteins with effector function are also suitable forcombination therapies and palliative regimes.

In one aspect, the present invention provides compositions and methodsuseful for treating or preventing diseases and conditions characterizedby aberrant levels of antigen activity associated with a cell. Thesediseases include cancers and other hyperproliferative conditions, suchas hyperplasia, psoriasis, contact dermatitis, immunological disorders,and infertility. A wide variety of cancers, including solid tumors andleukemias are amenable to the compositions and methods disclosed herein.Types of cancer that may be treated include, but are not limited to:adenocarcinoma of the breast, prostate, and colon; all forms ofbronchogenic carcinoma of the lung; myeloid; melanoma; hepatoma;neuroblastoma; papilloma; apudoma; choristoma; branchioma; malignantcarcinoid syndrome; carcinoid heart disease; and carcinoma (e.g.,Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor,Krebs 2, merkel cell, mucinous, non-small cell lung, oat cell,papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, andtransitional cell). Additional types of cancers that may be treatedinclude: histiocytic disorders; leukemia; histiocytosis malignant;Hodgkin's disease; immunoproliferative small; non-Hodgkin's lymphoma;plasmacytoma; reticuloendotheliosis; melanoma; chondroblastoma;chondroma; chondrosarcoma; fibroma; fibrosarcoma; giant cell tumors;histiocytoma; lipoma; liposarcoma; mesothelioma; myxoma; myxosarcoma;osteoma; osteosarcoma; chordoma; craniopharyngioma; dysgerminoma;hamartoma; mesenchymoma; mesonephroma; myosarcoma; ameloblastoma;cementoma; odontoma; teratoma; thymoma; trophoblastic tumor. Further,the following types of cancers are also contemplated as amenable totreatment: adenoma; cholangioma; cholesteatoma; cyclindroma;cystadenocarcinoma; cystadenoma; granulosa cell tumor; gynandroblastoma;hepatoma; hidradenoma; islet cell tumor; Leydig cell tumor; papilloma;sertoli cell tumor; theca cell tumor; leimyoma; leiomyosarcoma;myoblastoma; myomma; myosarcoma; rhabdomyoma; rhabdomyosarcoma;ependymoma; ganglioneuroma; glioma; medulloblastoma; meningioma;neurilemmoma; neuroblastoma; neuroepithelioma; neurofibroma; neuroma;paraganglioma; paraganglioma nonchromaffin. The types of cancers thatmay be treated also include, but are not limited to, angiokeratoma;angiolymphoid hyperplasia with eosinophilia; angioma sclerosing;angiomatosis; glomangioma; hemangioendothelioma; hemangioma;hemangiopericytoma; hemangiosarcoma; lymphangioma; lymphangiomyoma;lymphangiosarcoma; pinealoma; carcinosarcoma; chondrosarcoma;cystosarcoma phyllodes; fibrosarcoma; hemangiosarcoma; leiomyosarcoma;leukosarcoma; liposarcoma; lymphangiosarcoma; myosarcoma; myxosarcoma;ovarian carcinoma; rhabdomyosarcoma; sarcoma; neoplasms;nerofibromatosis; and cervical dysplasia. The invention further providescompositions and methods useful in the treatment of other conditions inwhich cells have become immortalized or hyperproliferative due toabnormally high expression of antigen.

Exemplifying the variety of hyperproliferative disorders amenable to thecompositions and methods of the invention are B-cell cancers, includingB-cell lymphomas (such as various forms of Hodgkin's disease,non-Hodgkins lymphoma (NHL) or central nervous system lymphomas),leukemias (such as acute lymphoblastic leukemia (ALL), chroniclymphocytic leukemia (CLL), Hairy cell leukemia and chronic myoblasticleukemia) and myelomas (such as multiple myeloma). Additional B cellcancers include small lymphocytic lymphoma, B-cell prolymphocyticleukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma,plasma cell myeloma, solitary plasmacytoma of bone, extraosseousplasmacytoma, extra-nodal marginal zone B-cell lymphoma ofmucosa-associated (MALT) lymphoid tissue, nodal marginal zone B-celllymphoma, follicular lymphoma, mantle cell lymphoma, diffuse largeB-cell lymphoma, mediastinal (thymic) large B-cell lymphoma,intravascular large B-cell lymphoma, primary effusion lymphoma,Burkitt's lymphoma/leukemia, B-cell proliferations of uncertainmalignant potential, lymphomatoid granulomatosis, and post-transplantlymphoproliferative disorder.

Disorders characterized by autoantibody production are often consideredautoimmune diseases. Autoimmune diseases include, but are not limitedto: arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis,osteoarthritis, polychondritis, psoriatic arthritis, psoriasis,dermatitis, polymyositis/dermatomyositis, inclusion body myositis,inflammatory myositis, toxic epidermal necrolysis, systemic sclerodermaand sclerosis, CREST syndrome, responses associated with inflammatorybowel disease, Crohn's disease, ulcerative colitis, respiratory distresssyndrome, adult respiratory distress syndrome (ARDS), meningitis,encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions,eczema, asthma, conditions involving infiltration of T cells and chronicinflammatory responses, atherosclerosis, autoimmune myocarditis,leukocyte adhesion deficiency, systemic lupus erythematosus (SLE),subacute cutaneous lupus erythematosus, discoid lupus, lupus myelitis,lupus cerebritis, juvenile onset diabetes, multiple sclerosis, allergicencephalomyelitis, neuromyelitis optica, rheumatic fever, Sydenham'schorea, immune responses associated with acute and delayedhypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis,sarcoidosis, granulomatosis including Wegener's granulomatosis andChurg-Strauss disease, agranulocytosis, vasculitis (includinghypersensitivity vasculitis/angiitis, ANCA and rheumatoid vasculitis),aplastic anemia, Diamond Blackfan anemia, immune hemolytic anemiaincluding autoimmune hemolytic anemia (AIHA), pernicious anemia, purered cell aplasia (PRCA), Factor VIII deficiency, hemophilia A,autoimmune neutropenia, pancytopenia, leukopenia, diseases involvingleukocyte diapedesis, central nervous system (CNS) inflammatorydisorders, multiple organ injury syndrome, myasthenia gravis,antigen-antibody complex mediated diseases, anti-glomerular basementmembrane disease, anti-phospholipid antibody syndrome, allergicneuritis, Behcet disease, Castleman's syndrome, Goodpasture's syndrome,Lambert-Eaton Myasthenic Syndrome, Reynaud's syndrome, Sjorgen'ssyndrome, Stevens-Johnson syndrome, solid organ transplant rejection,graft versus host disease (GVHD), bullous pemphigoid, pemphigus,autoimmune polyendocrinopathies, seronegative spondyloarthropathies,Reiter's disease, stiff-man syndrome, giant cell arteritis, immunecomplex nephritis, IgA nephropathy, IgM polyneuropathies or IgM mediatedneuropathy, idiopathic thrombocytopenic purpura (ITP), thromboticthrobocytopenic purpura (TTP), Henoch-Schonlein purpura, autoimmunethrombocytopenia, autoimmune disease of the testis and ovary includingautoimmune orchitis and oophoritis, primary hypothyroidism; autoimmuneendocrine diseases including autoimmune thyroiditis, chronic thyroiditis(Hashimoto's Thyroiditis), subacute thyroiditis, idiopathichypothyroidism, Addison's disease, Grave's disease, autoimmunepolyglandular syndromes (or polyglandular endocrinopathy syndromes),Type I diabetes also referred to as insulin-dependent diabetes mellitus(IDDM) and Sheehan's syndrome; autoimmune hepatitis, lymphoidinterstitial pneumonitis (HIV), bronchiolitis obliterans(non-transplant) vs NSIP, Guillain-Barré Syndrome, large vesselvasculitis (including polymyalgia rheumatica and giant cell (Takayasu's)arteritis), medium vessel vasculitis (including Kawasaki's disease andpolyarteritis nodosa), polyarteritis nodosa (PAN) ankylosingspondylitis, Berger's disease (IgA nephropathy), rapidly progressiveglomerulonephritis, primary biliary cirrhosis, Celiac sprue (glutenenteropathy), cryoglobulinemia, cryoglobulinemia associated withhepatitis, amyotrophic lateral sclerosis (ALS), coronary artery disease,familial Mediterranean fever, microscopic polyangiitis, Cogan'ssyndrome, Whiskott-Aldrich syndrome and thromboangiitis obliterans.

Rheumatoid arthritis (RA) is a chronic disease characterized byinflammation of the joints, leading to swelling, pain, and loss offunction. Patients having RA for an extended period usually exhibitprogressive joint destruction, deformity, disability and even prematuredeath. Beyond RA, inflammatory diseases, disorders and conditions ingeneral are amenable to treatment, prevention or amelioration ofsymptoms (e.g., heat, pain, swelling, redness) associated with theprocess of inflammation, and the compositions and methods of theinvention are beneficial in treating, preventing or amelioratingaberrant or abnormal inflammatory processes, including RA.

Crohn's disease and a related disease, ulcerative colitis, are the twomain disease categories that belong to a group of illnesses calledinflammatory bowel disease (IBD). Crohn's disease is a chronic disorderthat causes inflammation of the digestive or gastrointestinal (GI)tract. Although it can involve any area of the GI tract from the mouthto the anus, it most commonly affects the small intestine and/or colon.In ulcerative colitis, the GI involvement is limited to the colon.Crohn's disease may be characterized by antibodies against neutrophilantigens, i.e., the “perinuclear anti-neutrophil antibody” (pANCA), andSaccharomyces cervisiae, i.e. the “anti-Saccharomyces cerevisiaeantibody” (ASCA). Many patients with ulcerative colitis have the pANCAantibody in their blood, but not the ASCA antibody, while many Crohn'spatients exhibit ASCA antibodies, and not pANCA antibodies. One methodof evaluating Crohn's disease is using the Crohn's disease ActivityIndex (CDAI), based on 18 predictor variables scores collected byphysicians. CDAI values of 150 and below are associated with quiescentdisease; values above that indicate active disease, and values above 450are seen with extremely severe disease [Best et al., “Development of aCrohn's disease activity index.” Gastroenterology 70:439-444 (1976)].However, since the original study, some researchers use a ‘subjectivevalue’ of 200 to 250 as an healthy score.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease caused byrecurrent injuries to blood vessels in multiple organs, including thekidney, skin, and joints. In patients with SLE, a faulty interactionbetween T cells and B-cells results in the production of autoantibodiesthat attack the cell nucleus. There is general agreement thatautoantibodies are responsible for SLE, so new therapies that depletethe B-cell lineage, allowing the immune system to reset as new B-cellsare generated from precursors, would offer hope for long lasting benefitin SLE patients.

Multiple sclerosis (MS) is also an autoimmune disease. It ischaracterized by inflammation of the central nervous system anddestruction of myelin, which insulates nerve cell fibers in the brain,spinal cord, and body. Although the cause of MS is unknown, it is widelybelieved that autoimmune T cells are primary contributors to thepathogenesis of the disease. However, high levels of antibodies arepresent in the cerebral spinal fluid of patients with MS, and sometheories predict that the B-cell response leading to antibody productionis important for mediating the disease.

Autoimmune thyroid disease results from the production of autoantibodiesthat either stimulate the thyroid to cause hyperthyroidism (Graves'disease) or destroy the thyroid to cause hypothyroidism (Hashimoto'sthyroiditis). Stimulation of the thyroid is caused by autoantibodiesthat bind and activate the thyroid stimulating hormone (TSH) receptor.Destruction of the thyroid is caused by autoantibodies that react withother thyroid antigens.

Additional diseases, disorders, and conditions amenable to the benefitsprovided by the compositions and methods of the invention includeSjogren's syndrome is an autoimmune disease characterized by destructionof the body's moisture-producing glands. Further, immunethrombocytopenic purpura (ITP) is caused by autoantibodies that bind toblood platelets and cause their destruction, and this condition issuitable for application of the materials and methods of the invention.Myasthenia Gravis (MG), a chronic autoimmune neuromuscular disordercharacterized by autoantibodies that bind to acetylcholine receptorsexpressed at neuromuscular junctions leading to weakness of thevoluntary muscle groups, is a disease having symptoms that are treatableusing the composition and methods of the invention, and it is expectedthat the invention will be beneficial in treating and/or preventing MG.Still further, Rous Sarcoma Virus infections are expected to be amenableto treatment, or amelioration of at least one symptom, with thecompositions and methods of the invention.

Another aspect of the present invention is using the materials andmethods of the invention to prevent and/or treat any hyperproliferativecondition of the skin including psoriasis and contact dermatitis orother hyperproliferative disease. Psoriasis, is characterized byautoimmune inflammation in the skin and is also associated witharthritis in 30% of cases, as well as scleroderma, inflammatory boweldisease, including Crohn's disease and ulcerative colitis. It has beendemonstrated that patients with psoriasis and contact dermatitis haveelevated antigen activity within these lesions (Ogoshi et al., J. Inv.Dermatol., 110:818-23 [1998]). The multispecific, multivalent bindingproteins can deliver a cytotoxic cell of the immune system, for example,directly to cells within the lesions expressing high levels of antigen.The multivalent, e.g., multispecific, binding proteins can beadministered subcutaneously in the vicinity of the lesions, or by usingany of the various routes of administration described herein and otherswhich are well known to those of skill in the art.

Also contemplated is the treatment of idiopathic inflammatory myopathy(IIM), including dermatomyositis (DM) and polymyositis (PM).Inflammatory myopathies have been categorized using a number ofclassification schemes. Miller's classification schema (Miller, RheumDis Clin North Am. 20:811-826, 1994) identifies 2 idiopathicinflammatory myopathies (IIM), polymyositis (PM) and dermatomyositis(DM).

Polymyositis and dermatomyositis are chronic, debilitating inflammatorydiseases that involve muscle and, in the case of DM, skin. Thesedisorders are rare, with a reported annual incidence of approximately 5to 10 cases per million adults and 0.6 to 3.2 cases per million childrenper year in the United States (Targoff, Curr Probl Dermatol. 1991,3:131-180). Idiopathic inflammatory myopathy is associated withsignificant morbidity and mortality, with up to half of affected adultsnoted to have suffered significant impairment (Gottdiener et al., Am JCardiol. 1978, 41:1141-49). Miller (Rheum Dis Clin North Am. 1994,20:811-826 and Arthritis and Allied Conditions, Ch. 75, Eds. Koopman andMoreland, Lippincott Williams and Wilkins, 2005) sets out five groups ofcriteria used to diagnose IIM, i.e., Idiopathic Inflammatory MyopathyCriteria (IIMC) assessment, including muscle weakness, muscle biopsyevidence of degeneration, elevation of serum levels of muscle-associatedenzymes, electromagnetic triad of myopathy, evidence of rashes indermatomyositis, and also includes evidence of autoantibodies as asecondary criteria.

IIM associated factors, including muscle-associated enzymes andautoantibodies include, but are not limited to, creatine kinase (CK),lactate dehydrogenase, aldolase, C-reactive protein, aspartateaminotransferase (AST), alanine aminotransferase (ALT), and antinuclearautoantibody (ANA), myositis-specific antibodies (MSA), and antibody toextractable nuclear antigens.

Preferred autoimmune diseases amenable to the methods of the inventioninclude Crohn's disease, Guillain-Barré syndrome (GBS; also known asacute inflammatory demyelinating polyneuropathy, acute idiopathicpolyradiculoneuritis, acute idiopathic polyneuritis and Landry'sascending paralysis), lupus erythematosus, multiple sclerosis,myasthenia gravis, optic neuritis, psoriasis, rheumatoid arthritis,hyperthyroidism (e.g., Graves' disease), hypothyroidism (e.g.,Hashimoto's disease), Ord's thyroiditis (a thyroiditis similar toHashimoto's disease), diabetes mellitus (type 1), aplastic anemia,Reiter's syndrome, autoimmune hepatitis, primary biliary cirrhosis,antiphospholipid antibody syndrome (APS), opsoclonus myoclonus syndrome(OMS), temporal arteritis (also known as “giant cell arteritis”), acutedisseminated encephalomyelitis (ADEM), Goodpasture's syndrome, Wegener'sgranulomatosis, coeliac disease, pemphigus, canine polyarthritis, warmautoimmune hemolytic anemia. In addition, the invention contemplatesmethods for the treatment, or amelioration of a symptom associated with,the following diseases, endometriosis, interstitial cystitis,neuromyotonia, scleroderma, vitiligo, vulvodynia, Chagas' diseaseleading to Chagasic cardiopathy (cardiomegaly), sarcoidosis, chronicfatigue syndrome, and dysautonomia.

The complement system is believed to play a role in many diseases withan immune component, such as Alzheimer's disease, asthma, lupuserythematosus, various forms of arthritis, autoimmune heart disease andmultiple sclerosis, all of which are contemplated as diseases, disordersor conditions amenable to treatment or symptom amelioration using themethods according to the invention.

Certain constant sub-regions are preferred, depending on the particulareffector function or functions to be exhibited by a multivalentsingle-chain binding molecule. For example, IgG (IgG1, 2, or 3) and IgMare preferred for complement activation, IgG of any subtype is preferredfor opsonization and toxin neutralization; IgA is preferred for pathogenbinding; and IgE for binding of such parasites as worms.

By way of example, F_(C)Rs recognizing the constant region of IgGantibodies have been found on human leukocytes as three distinct typesof Fcγ receptors, which are distinguishable by structural and functionalproperties, as well as by antigenic structures detected by CD monoclonalantibodies. They are known as FcγRI, FcγRII, and FcγRIII, and aredifferentially expressed on (overlapping) subsets of leukocytes.

FcgRI (CD64), a high-affinity receptor expressed on monocytes,macrophages, neutrophils, myeloid precursors and dendritic cells,comprised isoforms 1a and 1b. FcgRI has a high affinity for monomerichuman IgG1 and IgG3. Its affinity for IgG4 is about 10 times lower,while it does not bind IgG2. FcgRI does not show genetic polymorphism.

FcγRII (CD32), comprised of isoforms 11a, 11b1, 11b2, 11b3 and 11c, isthe most widely distributed human FcγR type, being expressed on mosttypes of blood leukocytes, as well as on Langerhans cells, dendriticcells and platelets. FcγRII is a low-affinity receptor that only bindsaggregated IgG. It is the only FcγR class able to bind IgG2. FcγRIIashows genetics polymorphism, resulting in two distinct allotypes,FcγR11a-H131 and FcγR11a-R131, respectively. This functionalpolymorphism is attributable to a single amino acid difference: ahistidine (H) or an arginine (R) residue at position 131, which iscritical for IgG binding. FcγR11a readily binds human IgG and IgG3 andappears not to bind IgG4. The FcγR11a-H131 has a much higher affinityfor complexed IgG2 than the FcγR11a-R131 allotype.

FcγRIII (CD16) has two isoforms or allelotypes, both of which are ableto bind IgG1 and IgG3. The FcγRIIa, with an intermediate affinity forIgG, is expressed on macrophages, monocytes, natural killer (NK) cellsand subsets of T cells. FcγRIIIb is a low-affinity receptor for IgG,selectively expressed on neutrophils. It is a highly mobile receptorwith efficient collaboration with other membrane receptors. Studies withmyeloma IgG dimers have shown that only IgG1 and IgG3 bind to FcγRIIIb(with low affinity), while no binding of IgG2 and IgG4 has been found.The FcγRIIIb bears a co-dominant, bi-allelic polymorphism, the allotypesbeing designated NA1 (Neutrophil Antigen) and NA2.

Yet another aspect of the invention is use of the materials and methodsof the invention to combat, by treating, preventing or mitigating theeffects of, infection, resulting from any of a wide variety ofinfectious agents. The multivalent, multispecific binding molecules ofthe invention are designed to efficiently and effectively recruit thehost organism's immune system to resist infection arising from a foreignorganism, a foreign cell, a foreign virus or a foreign inanimate object.For example, a multispecific binding molecule may have one bindingdomain that specifically binds to a target on an infectious agent andanother binding domain that specifically binds to a target on an AntigenPresenting Cell, such as CD 40, CD80, CD86, DC-SIGN, DEC-205, CD83, andthe like). Alternatively, each binding domain of a multivalent bindingmolecule may specifically bind to an infectious agent, thereby moreeffectively neutralizing the agent. In addition, the inventioncontemplates multispecific, multivalent binding molecules thatspecifically bind to a target on an infectious agent and to anon-cell-associated binding partner, which may be effective inconjunction with an effector function of the multispecific bindingmolecule in treating or preventing infection arising from an infectiousagent.

Infectious cells contemplated by the invention include any knowninfectious cell, including but not limited to any of a variety ofbacteria (e.g., pathogenic E. coli, S. typhimurium, P. aeruginosa, B.anthracis, C. botulinum, C. difficile, C. perfringens, H. pylori, V.cholerae, and the like), mycobacteria, mycoplasma, fungi (includingyeast and molds), and parasites (including any known parasitic member ofthe Protozoa, Trematoda, Cestoda and Nematoda). Infectious virusesinclude, but are not limited to, eukaryotic viruses (e.g., adenovirus,bunyavirus, herpesvirus, papovavirus, paramyxovirus, picornavirus,poxvirus, reovirus, retroviruses, and the like) as well asbacteriophage. Foreign objects include objects entering an organism,preferably a human, regardless of mode of entry and regardless ofwhether harm is intended. In view of the increasing prevalence ofmulti-drug-resistant infectious agents (e.g., bacteria), particularly asthe causative agents of nosocomial infection, the materials and methodsof the invention, providing an approach to treatment that avoids thedifficulties imposed by increasing antibiotic resistance.

Diseases, conditions or disorders associated with infectious agents andamenable to treatment (prophylactic or therapeutic) with the materialsand methods disclosed herein include, but are not limited to, anthrax,aspergillosis, bacterial meningitis, bacterial pneumoniae (e.g.,chlamydia pneumoniae), blastomycosis, botulism, brucellosis,candidiasis, cholera, ciccidioidomycosis, cryptococcosis, diahhreagenic,enterohemorrhagic or enterotoxigenic E. coli, diphtheria, glanders,histoplasmosis, legionellosis, leprosy, listeriosis, nocardiosis,pertussis, salmonellosis, scarlet fever, sporotrichosis, strep throat,toxic shock syndrome, traveler's diarrhea, and typhoid fever.

Additional aspects and details of the invention will be apparent fromthe following examples, which are intended to be illustrative ratherthan limiting. Example 1 describes recombinant cloning of immunoglobulinheavy and light chain variable regions. Example 2 describes theconstruction of Small Modular ImmunoPharmaceuticals. Example 3 describesthe construction of a prototype cassette for a multivalent bindingprotein with effector function. Example 4 describes binding andexpression studies with this initial prototype molecule. Example 5describes construction of alternative constructs derived from thisinitial prototype molecule where the sequence of the linker regionbetween the EFD and BD2 was changed in both length and sequence. Inaddition, it describes alternative forms where the orientation of Vregions in binding domain 2 were also altered. Example 6 describessubsequent binding and functional studies on these alternativeconstructs with variant linker forms, identifying a cleavage in thelinker region in several of these derivative forms, and the new sequencevariants developed to address this problem. Example 7 describes theconstruction of an alternative preferred embodiment of themultispecific, multivalent fusion proteins, where both BD1 and BD2 bindto antigens on the same cell type (CD20 and CD37), or anothermultispecific fusion protein where the antigen binding specificity forBD2 has been changed to human CD3 instead of CD28. Example 8 describesthe binding and functional studies performed with the CD20-hIgG-CD37multispecific constructs. Example 9 describes the binding and functionalstudies with the CD20-hIgG-CD3 multivalent fusion protein constructs.Example 10 discloses multivalent binding molecules having linkers basedon specific regions of the extracellular domains of members of theimmunoglobulin superfamily. Example 11 discloses assays for identifyingbinding domains expected to be effective in multivalent bindingmolecules in achieving at least one beneficial effect identified asbeing associated with such molecules (e.g., disease treatment).

Example 1 Cloning of Immunoglobulin Heavy and Light Chain VariableRegions

Any methods known in the art can be used to elicit antibodies to a givenantigenic target. Further, any methods known in the art can be used toclone the immunoglobulin light and/or heavy chain variable regions, aswell as the constant sub-region of an antibody or antibodies. Thefollowing method provides an exemplary cloning method.

A. Isolation of Total RNA

To clone the immunoglobulin heavy and light chain variable regions, orthe constant sub-region, total RNA is isolated from hybridoma cellssecreting the appropriate antibody. Cells (2×10⁷) from the hybridomacell line are washed with 1×PBS and pelleted via centrifugation in a12×75 mm round bottom polypropylene tube (Falcon no. 2059). TRIzol™Total RNA Isolation Reagent (Gibco BRL, Life Technologies, Cat no.15596-018) is added (8 ml) to each tube and the cells are lysed viarepeated pipetting. The lysate is incubated for 5 minutes at roomtemperature prior to the addition of 1.6 ml (0.2× volume) of chloroformand vigorous shaking for 15 seconds. After standing 3 minutes at roomtemperature, the lysates are centrifuged at 9,000 rpm for 15 minutes ina 4° C. pre-chilled Beckman JA-17 rotor in order to separate the aqueousand organic phases. The top aqueous phase (about 4.8 ml) is transferredinto a new tube and mixed gently with 4 ml of isopropanol. After a 10minute incubation at room temperature, the RNA is precipitated bycentrifugation at 9,000 rpm in a 4° C. JA-17 rotor for 11 minutes. TheRNA pellet is washed with 8 ml of ice-cold 75% ethanol and re-pelletingby centrifugation at 7,000× rpm for 7 minutes in a JA-17 rotor at 4° C.The ethanol wash is decanted and the RNA pellets are air-dried for 10minutes. The RNA pellets are resuspended in 150 μl ofdiethylpyrocarbonate (DEPC)-treated ddH₂O containing 1 μl of RNaseInhibitor (Catalog No. 799017; Boehringer Mannheim/Roche) per 1 ml ofDEPC-treated ddH₂O. The pellets are resuspended by gentle pipetting andare incubated for 20 minutes at 55° C. RNA samples are quantitated bymeasuring the OD_(260 nm) of diluted aliquots (1.0 OD_(260 nm) unit=40μg/ml RNA).

B. Rapid Amplification of cDNA Ends

5′ RACE is carried out to amplify the ends of the heavy and light chainvariable regions, or the constant sub-region. The 5′ RACE System forRapid Amplification of cDNA Ends Kit version 2.0 (Life Technologies,cat. no. 18374-058) is used according to the manufacturer'sinstructions. Degenerate 5′ RACE oligonucleotide primers are designed tomatch, e.g., the constant regions of two common classes of mouseimmunoglobulin heavy chains (IgG1 and IgG2b) using the oligonucleotidedesign program Oligo version 5.1 (Molecular Biology Insights, CascadeColo.). Primers are also designed to match the constant region of themouse IgG kappa light chain. This is the only class of immunoglobulinlight chain, so no degeneracy is needed in the primer design. Thesequences of the primers are as follows:

Name Sequence SEQ ID NO Heavy Chain GSPI 75′AGGTGCTGGAGGGGACAGTCACTGAGCTGC3′ Nested Heavy Chain 85′GTCACWGTCACTGRCTCAGGGAARTAGC3′ (W = A or T; R = A or G) Light ChainGSP1 9 5′GGGTGCTGCTCATGCTGTAGGTGCTGTCTTTGC3′ Nested Light Chain 105′CAAGAAGCACACGACTG AGGCACCTCCAGATG3′ 5′ Race Abridged Anchor Primer 115′GGCCACGCGTCGACTAGTACGG GNNGGGNNGGGNNG3′

To amplify the mouse immunoglobulin heavy chain component, the reversetranscriptase reaction is carried in a 0.2 ml thin-walled PCR tubecontaining 2.5 pmoles of heavy chain GSP1 primer (SEQ ID NO: 7), 4 μg oftotal RNA isolated from a suitable hybridoma clone (e.g., either clone4A5 or clone 4B5), and 12 μl of DEPC treated ddH2O. Likewise, for themouse light chain component, the reverse transcriptase reaction iscarried out in a 0.2 ml thin-walled PCR tube containing 2.5 pmoles of alight chain GSP1 primer (SEQ ID NO: 9), 4 μg of total RNA from asuitable hybridoma clone (e.g., either clone 4A5 or clone 4B5), and 12μl of DEPC treated ddH2O.

The reactions are carried out in a PTC-100 programmable thermal cycler(MJ research Inc., Waltham, Mass.). The mixture is incubated at 70° C.for 10 minutes to denature the RNA and then chilled on wet ice for 1minute. The tubes are centrifuged briefly in order to collect moisturefrom the lids of the tubes. Subsequently, the following components areadded to the reaction: 2.5 μl of 10×PCR buffer (200 mM Tris-HCl, pH 8.4,500 mM KCl), 2.5 μl of 25 mM MgCl₂, 1 μl of 10 mM dNTP mix, and 2.5 μlof 0.1 M DTT. After mixing each tube by gentle pipetting, the tubes areplaced in a PTC-100 thermocycler at 42° C. for 1 minute to pre-warm themix. Subsequently, 1 μl (200 units) of SuperScript™ II ReverseTranscriptase (Gibco-BRL; cat no. 18089-011) is added to each tube,gently mixed by pipetting, and incubated for 45 minutes at 42° C. Thereactions are cycled to 70° C. for 15 minutes to terminate the reaction,and then cycled to 37° C. RNase mix (1 μl) is then added to eachreaction tube, gently mixed, and incubated at 37° C. for 30 minutes.

The first-strand cDNA generated by the reverse transcriptase reaction ispurified with the GlassMAX DNA Isolation Spin Cartridge (Gibco-BRL)according to the manufacturer's instructions. To each first-strandreaction, 120 μl of 6 M NaI binding solution is added. The cDNA/NaIsolution is then transferred into a GlassMAX spin cartridge andcentrifuged for 20 seconds at 13,000×g. The cartridge inserts arecarefully removed and the flow-through is discarded from the tubes. Thespin cartridges are then placed back into the empty tubes and 0.4 ml ofcold (4° C.) 1×wash buffer is added to each spin cartridge. The tubesare centrifuged at 13,000×g for 20 seconds and the flow-through isdiscarded. This wash step is repeated three additional times. TheGlassMAX cartridges are then washed 4 times with 0.4 ml of cold (4° C.)70% ethanol. After the flow-through from the final 70% ethanol wash isdiscarded, the cartridges are placed back in the tubes and centrifugedat 13,000×g for an additional 1 minute in order to completely dry thecartridges. The spin cartridge inserts are then transferred to a freshsample recovery tube where 50 μl of 65° C. (pre-heated) DEPC-treatedddH₂O is quickly added to each spin cartridge. The cartridges arecentrifuged at 13,000×g for 30 seconds to elute the cDNA.

C. Terminal Deoxynucleotidyl Transferase (TdT) Tailing

For each first-strand cDNA sample, the following components are added toa 0.2 ml thin-walled PCR tube: 6.5 μl of DEPC-treated ddH₂O, 5.0 μl of5× tailing buffer, 2.5 μl of 2 mM dCTP, and 10 μl of the appropriateGlassMAX-purified cDNA sample. Each 24 μl reaction is incubated 2-3minutes in a thermal cycler at 94° C. to denature the DNA, and chilledon wet ice for 1 minute. The contents of the tube are collected by briefcentrifugation. Subsequently, 1 μl of terminal deoxynucleotidyltransferase (TdT) is added to each tube. The tubes are mixed via gentlepipetting and incubated for 10 minutes at 37° C. in a PTC-100 thermalcycler. Following this 10 minute incubation, the TdT is heat inactivatedby cycling to 65° C. for 10 minutes. The reactions are cooled on ice andthe TdT-tailed first-strand cDNA is stored at −20° C.

D. PCR of dC-tailed First-Strand cDNA

Duplicate PCR amplifications (two independent PCR reactions for eachdC-tailed first-strand cDNA sample) are performed in a 50 μl volumecontaining 200 μM dNTPs, 0.4 μM of 5′ RACE Abridged Anchor Primer (SEQID NO: 11), and 0.4 μM of either Nested Heavy Chain GSP2 (SEQ ID NO: 8)or Nested Light Chain GSP2 (SEQ ID NO: 10), 10 mM Tris-HCl (pH 8.3), 1.5mM MgCl₂, 50 mM KCl, 5 μl of dC-tailed cDNA, and 5 units of Expand™Hi-Fi DNA polymerase (Roche/Boehringer Mannheim GmbH, Germany). The PCRreactions are amplified using a “Touch-down/Touch-up” annealingtemperature protocol in a PTC-100 programmable thermal cycler (MJResearch Inc.) with the following conditions: initial denaturation of95° C. for 40 seconds, 5 cycles at 94° C. for 20 seconds, 61° C.-2°C./cycle for 20 seconds, 72° C. for 40 seconds+1 second/cycle, followedby 5 cycles at 94° C. for 25 seconds, 53° C.+1° C./cycle for 20 seconds,72° C. for 46 seconds+1 second/cycle, followed by 20 cycles at 94° C.for 25 seconds, 55° C. for 20 seconds, 72° C. for 51 seconds+1second/cycle, and a final incubation of 72° C. for 5 minutes.

E. TOPO TA-Cloning

The resulting PCR products are gel-purified from a 1.0% agarose gelusing the QIAQuick Gel purification system (QIAGEN Inc., Chatsworth,Calif.), TA-cloned into pCR2.1 using the TOPO TA Cloning® kit(Invitrogen, San Diego, Calif., cat. no. K4550-40), and transformed intoE. coli TOP10F′ cells (Invitrogen), according to manufacturers'instructions. Clones with inserts are identified by blue/white screeningaccording to the manufacturer's instructions, where white clones areconsidered positive clones. Cultures of 3.5 ml liquid Luria Broth (LB)containing 50 μg/ml ampicillin are inoculated with white colonies andgrown at 37° C. overnight (about 16 hours) with shaking at 225 rpm.

The QIAGEN Plasmid Miniprep Kit (QIAGEN Inc., cat. no. 12125) is used topurify plasmid DNA from the cultures according to the manufacturer'sinstructions. The plasmid DNA is suspended in 34 μl of 1×TE buffer (pH8.0) and then positive clones sequenced as previously described byfluorescent dideoxy nucleotide sequencing and automated detection usingABI Big Dye Terminator 3.1 reagents at 1:4-1:8 dilutions and analyzedusing an ABI 3100 DNA sequencer. Sequencing primers used include T7(5′GTAATACGACTCACTATAGG3′; SEQ ID NO: 12) and M13 Reverse(5′CAGGAAACAGCTATGACC3′; SEQ ID NO: 13) primers. Sequencing results willconfirm that the clones correspond to mouse IgG sequences.

F. De Novo Gene Synthesis Using Overlapping Oligonucleotide ExtensionPCR

This method involves the use of overlapping oligonucleotide primers andPCR using either a high fidelity DNA polymerase or a mix of polymerasesto synthesize an immunoglobulin V-region or other gene. Starting at themiddle of the V-region sequence, 40-50 base primers are designed suchthat the growing chain is extended by 20-30 bases, in either direction,and contiguous primers overlap by a minimum of 20 bases. Each PCR steprequires two primers, one priming on the anti-sense strand (forward orsense primer) and one priming on the sense strand (reverse or anti-senseprimer) to create a growing double-stranded PCR product. During primerdesign, changes can be made in the nucleotide sequence of the finalproduct to create restriction enzyme sites, destroy existing restrictionenzyme sites, add flexible linkers, change, delete or insert bases thatalter the amino acid sequence, optimize the overall DNA sequence toenhance primer synthesis and conform to codon usage rules for theorganism contemplated for use in expressing the synthetic gene.

Primer pairs are combined and diluted such that the first pair are at 5μM an each subsequent pair has a 2-fold greater concentration up to 80μM. One μL from each of these primer mixes is amplified in a 50 μL PCRreaction using Platinum PCR SuperMix-High Fidelity (Invitrogen, SanDiego, Calif., cat. no. 12532-016). After a 2-minute initialdenaturation at 94° C., 30 cycles of PCR are performed using a cyclingprofile of 94° C. for 20 seconds, 60° C. for 10 seconds; and 68° C. for15 seconds. PCR products are purified using Qiaquick PCR Purificationcolumns (Qiagen Inc., cat. no. 28704) to remove excess primers andenzyme. This PCR product is then reamplified with the next set ofsimilarly diluted primer pairs using PCR conditions exactly as describedabove, but increasing the extension time of each cycle to 68° C. for 30seconds. The resultant PCR product is again purified from primers andenzymes as described above and TOPO-TA cloned and sequenced exactly asdescribed in section E above.

Example 2 Construction of Small Modular ImmunoPharmaceuticals (SMIPs)

A multispecific, multivalent binding protein with effector function wasconstructed that contained a binding domain 1 in the form of asingle-chain recombinant (murine/human) scFv designated 2H7(VL-linker-VH). The scFv 2H7 is a small modular immunopharmacaceutical(SMIP) that specifically recognizes CD20. The binding domain was basedon a publicly available human CD20 antibody sequence GenBank AccessionNumbers, M17953 for VH, and M17954 for VL. CD20-specific SMIPs aredescribed in co-owned US Patent Publications 2003/133939, 2003/0118592and 2005/0136049, incorporated herein in their entireties by reference.The peptide linker separating VL and VH was a 15-amino acid linkerencoding the sequence: Asp-Gly₃Ser-(Gly₄Ser)₂. Binding domain 1 waslocated at the N-terminus of the multispecific binding protein, with theC-terminus of that domain linked directly to the N-terminus of aconstant sub-region containing a hinge, C_(H2) and C_(H3) domains (inamino-to-carboxy orientation). The constant sub-region was derived froman IgG1 antibody, which was isolated by PCR amplification of human IgG1from human PBMCs. The hinge region was modified by substituting threeSer residues in place of the three Cys residues present in the wild typeversion of the human IgG1 hinge domain, encoded by the 15 amino acidsequence: EPKSCDKTHTCPPCP (SEQ ID NO: 14; the three Cys residuesreplaced by Ser residues are indicated in bold). In alternativeembodiments, the hinge region was modified at one or more of thecysteines, so that SSS and CSC type hinges were generated. In addition,the final proline was sometimes substituted with a serine as well as thecysteine substitutions.

The C-terminal end of the C_(H3) domain was covalently attached to aseries of alternative linker domains juxtaposed between the constantsub-region C-terminus and the amino terminus of binding domain 2.Preferred multivalent binding proteins with effector function will haveone of these linkers to space the constant sub-region from bindingdomain 2, although the linker is not an essential component of thecompositions according to the invention, depending on the foldingproperties of BD2. For some specific multivalent molecules, the linkermight be important for separation of domains, while for others it may beless important. The linker was attached to the N-terminal end of scFv2E12 ((V_(H)-linker-V_(L)), which specifically recognizes CD28. Thelinker separating the VH and VL domains of the scFv 2E12 part of themultivalent binding molecule was a 20-amino acid linker (Gly₄Ser)₄,rather than the standard (Gy₄Ser)₃ linker usually inserted between Vdomains of an scFv. The longer linker was observed to improve thebinding properties of the 2e12 scFv in the VH-VL orientation.

The multispecific, multivalent binding molecule as constructed containeda binding domain 1, which comprises the 2E12 leader peptide sequencefrom amino acids 1-23 of SEQ ID NO: 171; the 2H7 murine anti-human CD20light chain variable region, which is reflected at position 24 in SEQ IDNO: 171; an Asp-Gly₃-Ser-(Gly₄Ser)₂ linker, beginning at residue 130 inSEQ ID NO: 171, the 2H7 murine anti-human CD20 heavy chain variableregion with a leucine to serine (VHL11S) amino acid substitution atresidue 11 in the variable domain for VH, and which has a single serineresidue at the end of the heavy chain region (i.e., VTVS where acanonical sequence would be VTVSS) (Genbank Acc. No. M17953), andinterposed between the two binding domains BD1 (2H7) and BD2 (2E12) is ahuman IgG1 constant sub-region, including a modified hinge regioncomprising a “CSC” or an “SSS” sequence, and wild-type C_(H2) and C_(H3)domains. The nucleotide and amino acid sequences of the multivalentbinding protein with effector function are set out in SEQ ID NOS: 228and 229 for the CSC forms, respectively and SEQ ID NOS: 170 and 171, forthe SSS forms.

Stably expressing cell lines were created by transfection viaelectroporation of either uncut or linearized, recombinant expressionplasmid into Chinese hamster ovary cells (CHO DG44 cells) followed byselection in methotrexate containing medium. Bulk cultures and masterwells producing the highest level of multivalent binding protein wereamplified in increasing levels of methotrexate, and adapted cultureswere subsequently cloned by limiting dilution. Transfected CHO cellsproducing the multivalent binding protein were cultured in bioreactorsor wave bags using serum-free medium obtained from JRH Biosciences(Excell 302, cat. no. 14324-1000M, supplemented with 4 mM glutamine(Invitrogen, 25030-081), sodium pyruvate (Invitrogen 11360-070, dilutedto 1X), non-essential amino acids (Invitrogen, 11140-050, final dilutionto 1×), penicillin-streptromycin 100 IU/ml (Invitrogen, 15140-122), andrecombulin insulin at 1 μg/ml (Invitrogen, 97-503311). Other serum freeCHO basal medias may also be used for production, such as CD-CHO, andthe like.

Fusion protein was purified from spent CHO culture supernatants byProtein A affinity chromatography. The multivalent binding protein waspurified using a series of chromatography and filtration steps,including a virus reduction filter. Cell culture supernatants werefiltered, then subjected to protein A Sepharose affinity chromatographyover a GE Healthcare XK 16/40 column. After binding of protein to thecolumn, the column was washed in dPBS, then 1.0 M NaCl, 20 mM sodiumphosphate pH 6.0, and then 25 mM NaCl, 25 mN NaOAc, pH 5.0 to removenonspecific binding proteins. Bound protein was eluted from the columnin 100 mM Glycine (Sigma), pH 3.5, and brought to pH 5.0 with 0.5 M2-(N-Morpholino) ethanesulfonic acid (MES), pH 6.0. Protein samples wereconcentrated to 25 mg/ml in preparation for GPC purification. Sizeexclusion chromatography was performed on a GE Healthcare AKTA Explorer100 Air apparatus, using a GE healthcare XK column and Superdex 200preparative grade (GE healthcare).

The material was then concentrated and formulated with 20 mM sodiumphosphate and 240 mM sucrose, with a resulting pH of 6.0. Thecomposition was filtered before filling into sterile vials at variousconcentrations, depending on the amount of material recovered.

Example 3 Construction of Scorpion Expression Cassette

A nucleic acid containing the synthetic 2H7 scFv (anti-CD20; SEQ IDNO: 1) linked to a constant sub-region as described in Example 2 hasbeen designated TRU-015. TRU-015 nucleic acid, as well as synthetic scFv2E12 (anti-CD28 VL-VH; SEQ ID NO: 3) and synthetic scFv 2E12 (anti-CD28VH-VL; SEQ ID NO: 5) nucleic acids encoding small modularimmunopharmaceuticals, were used as templates for PCR amplification ofthe various components of the scorpion cassettes The template, orscaffold, for binding domain 1 and the constant sub-region was providedby TRU-015 (the nucleic acid encoding scFv 2H7 (anti-CD20) linked to theconstant sub-region) and this template was constructed in the expressionvector pD18. The above-noted nucleic acids containing scFv 2E12 ineither of two orientations (V_(L)-V_(H) and V_(H)-V_(L)) provided thecoding region for binding domain 2.

TRU 015 SSS Hinge C_(H2)C_(H3) for BD2/Linker Insertion

A version of the synthetic 2H7 scFv IgG1 containing the SSS hinge wasused to create a scorpion cassette by serving as the template foraddition of an EcoRI site to replace the existing stop codon and XbaIsite. This molecule was amplified by PCR using primer 9 (SEQ ID NO: 23;see Table 1) and primer 87 (SEQ ID NO: 40; see Table 1) as well as aPlatinum PCR High Fidelity mix (Invitrogen). The resultant 1.5 Kbpfragment was purified and cloned into the vector pCR2.1-TOPO(Invitrogen), transformed into E. coli strain TOP10 (Invitrogen), andthe DNA sequence verified.

TABLE 1 Table 1. Oligonucleotide primers used to constructCD20-CD28 scorpion cassette. Primers areseparated into 2 groups, PCR and Sequencing.PCR primers were used to construct the cassetteand sequencing primers were used to confirm theDNA sequence of all intermediates and final constructs. SEQ ID No. NameSequence 5′-3′ NO. PCR Primers 1 hVK3L-F3H3 GCGATAAAGCTTGCCGCCATGGAA 15GCACCAGCGCAGCTTCTCTTCC 2 hVK3L-F2 ACCAGCGCAGCTTCTCTTCCTCCTG 16CTACTCTGGCTCCCAGATACCACCG 3 hVK3L-F1- GGCTCCCAGATACCACCGGTCAAAT 17 2H7VLTGTTCTCTCCCAGTCTCCAG 4 2H7VH-NheF GCGATAGCTAGCCAGGCTTATCTAC 18AGCAGTCTGG 5 G4S-NheR GCGATAGCTAGCCCCACCTCCTCCA 19 GATCCACCACCGCCCGAG 6015VH-XhoR GCGTACTCGAGGAGACGGTGACCGT 20 GGTCCCTGTG 7 G1H-C-XHOGCAGTCTCGAGCGAGCCCAAATCTTG 21 TGACAAAACTC 8 G1H-S-XHOGCAGTCTCGAGCGAGCCCAAATCTTC 22 TGACAAAACTC 9 CH3R-EcoR1GCGTGAGAATTCTTACCCGGAGACAGG 23 GAGAGGCTC 10 G1-XBA-RGCGACGTCTAGAGTCATTTACCCGGAG 24 ACAGG 11 G4SLinkR1-SAATTATGGTGGCGGTGGCTCGGGCGGT 25 GGTGGATCTGGAGGAGGTGGGAGTGGG 12G4SLinkR1-AS AATTCCCACTCCCACCTCCTCCAGATCCA 26 CCACCGCCCGAGCCACCGCCACCAT13 2E12VLXbaR GCGTGTCTAGATTAACGTTTGATTTCCAG 27 CTTGGTG 14 2E12VLR1FGCGATGAATTCTGACATTGTGCTCACCCA 28 ATCTCC 15 2E12VHR1FGCGATGAATTCTCAGGTGCAGCTGAAGGA 29 GTCAG 16 2E12VHXbaRGCGAGTCTAGATTAAGAGGAGACGGTGAC 30 TGAGGTTC 17 2e12VHdXbaF1GGGTCTGGAGTGGCTGGGAATGATATG 31 18 2e12VHdXbaR1ATTCCCAGCCACTCCAGACCCTTTCCTG 32 19 IgBsrG1F GAGAACCACAGGTGTACACCCTG 3320 IgBsrG1R GCAGGGTGTACACCTGTGGTTCTCG 34 Sequencing Primers 82 M13RCAGGAAACAGCTATGAC 35 83 M13F GTAAAACGACGGCCAGTG 36 84 T7GTAATACGACTCACTATAGG 37 85 pD18F-17 AACTAGAGAACCCACTG 38 86 pD18F-20GCTAACTAGAGAACCCACTG 39 87 pD18F-1 ATACGACTCACTATAGGG 40 88 pD18R-sGCTCTAGCATTTAGGTGAC 41 89 CH3seqF1 CATGAGGCTCTGCACAAC 42 90 CH3seqF2CCTCTACAGCAAGCTCAC 43 91 CH3seqR1 GGTTCTTGGTCAGCTCATC 44 92 CH3seqR2GTGAGCTTGCTGTAGAGG 45n2H7 V_(K) and Human V_(K3) Leader Sequence Fusion

Oligonucleotide-directed PCR mutagenesis was used to introduce an AgeI(ACCGGT) restriction site at the 5′ end of the coding region for TRU 015VK and an Nhe I (GCTAGC) restriction site at the 3′ end of the codingregion for the (G4S)3 linker using primers 3 and 5 from Table 1. Sinceprimer 3 also encodes the last 6 amino acids of the human VK3 leader(gb:X01668), overlapping PCR was used to sequentially add the N-terminalsequences of the leader including a consensus Kozak box and HinDIII(AAGCTT) restriction site using primers 1, 2 and 5 from Table 1.

n2H7 IgG1 SSS Hinge-C_(H2)C_(H3) Construction

Primers 4 and 6 (SEQ ID NOS: 18 and 20, respectively; Table 1) were usedto re-amplify the TRU-015 V_(H) with an NheI site 5′ to fuse with theV_(K) for TRU-015 and an Xho I (5′-CTCGAG-3′) site at the 3′ endjunction with the IgG1 hinge-C_(H2)C_(H3) domains. Likewise, the IgG1hinge-C_(H2)-C_(H3) region was amplified using primers 8 and 9 fromTable 1, introducing a 5′ Xho I site and replacing the existing 3′ endwith an EcoRI (5′-GAATTC-3′) site for cloning, and destroying the stopcodon to allow translation of Binding Domain 2 attached downstream ofthe CH3 domain. This version of the scorpion cassette is distinguishedfrom the previously described cassette by the prefix “n.”

In addition to the multivalent binding protein described above, aprotein according to the invention may have a binding domain, eitherbinding domain 1 or 2 or both, that corresponds to a single variableregion of an immunoglobulin. Exemplary embodiments of this aspect of theinvention would include binding domains corresponding to the V_(H)domain of a camelid antibody, or a single modified or unmodified Vregion of another species antibody capable of binding to the targetantigen, although any single variable domain is contemplated as usefulin the proteins of the invention.

2E12 VL-VH and VH-VL Constructions

In order to make the 2E12 scFvs compatible with the cassette, aninternal Xba I (5′-TCTAGA-3′) site had to be destroyed using overlappingoligonucleotide primers 17 and 18 from Table 1. These two primers incombination with primer pairs 14/16 (VL-VH) or 13/15 (VH-VL) were usedto amplify the two oppositely oriented binding domains such that theyboth carried EcoRI and XbaI sites at their 5′ and 3′ ends, respectively.Primers 13 and 16 also encode a stop codon (TAA) immediately in front ofthe Xba I site.

2H7 SSS IgG12e12 LH/HL Construction Effector Domain-Binding Domain 2Linker Addition. (STD Linkers—STD1 and STD2)

Complementary primers 11 and 12 from Table 1 were combined, heated to70° C. and slow-cooled to room temperature to allow annealing of the twostrands. 5′ phosphate groups were added using T4 polynucleotide kinase(Roche) in 1× Ligation buffer with 1 mM ATP (Roche) using themanufacturer's protocol. The resulting double-stranded linker was thenligated into the EcoRI site between the coding regions for the IgG1C_(H3) terminus and the beginning of Binding Domain 2 using T4 DNAligase (Roche). The resultant DNA constructs were screened for thepresence of an EcoRI site at the linker-BD2 junction and the nucleotidesequence GAATTA at the C_(H3)-linker junction. The correct STD 1 linkerconstruct was then re-digested with EcoRI and the linker ligationrepeated to produce a molecule that had a linker composed of two (STD 2)identical iterations of the Lx1 sequence. DNA constructs were againscreened as above.

Example 4 Expression Studies

Expression studies were performed on the nucleic acids described abovethat encode multivalent binding proteins with effector function. Nucleicacids encoding multivalent binding proteins were transiently transfectedinto COS cells and the transfected cells were maintained under wellknown conditions permissive for heterologous gene expression in thesecells. DNA was transiently transfected into COS cells using PEI orDEAE-Dextran as previously described (PEI=Boussif O. et al., PNAS 92:7297-7301, (1995), incorporated herein by reference; Pollard H. et al.,JBC 273: 7507-7511, (1998), incorporated herein by reference). Multipleindependent transfections of each new molecule were performed in orderto determine the average expression level for each new form. Fortransfection by PEI, COS cells were plated onto 60 mm tissue cultureplates in DMEM/10% FBS medium and incubated overnight so that they wouldbe approximately 90% confluent on the day of transfection. Medium waschanged to serum free DMEM containing no antibiotics and incubated for 4hours. Transfection medium (4 ml/plate) contained serum free DMEM with50 μg PEI and 10-20 ug DNA plasmid of interest. Transfection medium wasmixed by vortexing, incubated at room temperature for 15 minutes, andadded to plates after aspirating the existing medium. Cultures wereincubated for 3-7 days prior to collection of supernatants. Culturesupernatants were assayed for protein expression by SDS-PAGE, Westernblotting, binding verified by flow cytometry, and function assayed usinga variety of assays including ADCC, CDC, and coculture experiments.

SDS-PAGE Analysis and Western Blotting Analysis

Samples were prepared either from crude culture supernatants (usually 30μl/well) or purified protein aliquots, containing 8 ug protein per well,and 2× Tris-Glycine SDS Buffer (Invitrogen) was added to a 1× finalconcentration. Ten (10) μl SeeBlue Marker (Invitrogen, Carlsbad, Calif.)were run to provide MW size standards. The multivalent binding (fusion)protein variants were subjected to SDS-PAGE analysis on 4-20% NovexTris-glycine gels (Invitrogen, San Diego, Calif.). Samples were loadedusing Novex Tris-glycine SDS sample buffer (2×) under reducing ornon-reducing conditions after heating at 95° C. for 3 minutes, followedby electrophoresis at 175V for 60 minutes. Electrophoresis was performedusing 1× Novex Tris-Glycine SDS Running Buffer (Invitrogen).

After electrophoresis, proteins were transferred to PVDF membranes usinga semi-dry electroblotter apparatus (Ellard, Seattle, Wash.) for 1 hourat 100 mAmp. Western transfer buffers included the following threebuffers present on saturated Whatman filter paper, and stacked insuccession: no. 1 contains 36.34 g/liter Tris, pH 10.4, and 20%methanol; no. 2 contains 3.02 g/liter Tris, pH 10.4, and 20% methanol;and no. 3 contains 3.03 g/liter Tris, pH 9.4, 5.25 g/liter ε-aminocaproic acid, and 20% methanol. Membranes were blocked in BLOTTO=5%nonfat milk in PBS overnight with agitation. Membranes were incubatedwith HRP conjugated goat anti-human IgG (Fc specific, Caltag) at 5 ug/mlin BLOTTO for one hour, then washed 3 times for 15 minutes each inPBS-0.5% Tween 20. Wet membranes were incubated with ECL solution for 1minute, followed by exposure to X-omat film for 20 seconds. FIG. 2 showsa Western Blot of proteins expressed in COS cell culture supernatant (30μl/well) electrophoresed under non-reducing conditions. Lanes areindicated with markers 1-9 and contain the following samples: Lane 1(cut off=See Blue Markers, kDa are indicated to the side of the blot.Lane 2=2H7-sssIgG P238S/P331S-STD1-2e12 VLVH; lane 3=2H7-sssIgGP238S/P331S-STD1-2e12 VHVL, Lane 4=2H7-sssIgG P238S/P331S-STD2-2e12VLVH; Lane 5=2H7-sssIgG P238S/P331S-STD2-2e12 VHVL; Lane 6=2e12 VLVHSMIP; Lane 7=2e12 VHVL SMIP; Lane 8=2H7 SMIP. 2H7 in these constructs isalways in the V_(L)V_(H) orientation, sssIgG indicates the identity ofthe hinge/linker located at linker position 1 as shown in FIG. 5,P238S/P331S indicates the version of human IgG1 with mutations from wildtype (first aa listed) to mutant (second aa listed) and the amino acidposition at which they occur in wild type human IgG1 C_(H2) and CH3domains, STD1 indicates the 20-amino-acid (18+restriction site) linkerlocated in linker position 2 as shown in FIG. 5, and STD2 indicates the38 amino acid (36+restriction site) linker located in linker position 2as shown in FIG. 6.

Binding Studies

Binding studies were performed to assess the bispecific bindingproperties of the CD20/CD28 multispecific, multivalent binding peptides.Initially, WIL2-S cells were added to 96 well plates and centrifuged topellet cells. To the seeded plates, CD20/CD28 purified protein wasadded, using two-fold titrations across the plate from 20 μg/ml down to0.16 μg/ml. A two-fold dilution series of TRU-015 (source of bindingdomain 1) purified protein was also added to seeded plate wells, theconcentration of TRU-015 extending from 20 μg/ml down to 0.16 μg/ml. Onewell containing no protein served as a background control.

Seeded plates containing the proteins were incubated on ice for onehour. Subsequently, the wells were washed once with 200 μl 1% FBS inPBS. Goat anti-human antibody labeled with FITC (Fc Sp) at 1:100 wasthen added to each well, and the plates were again incubated on ice forone hour. The plates were then washed once with 200 μl 1% FBS in PBS andthe cells were re-suspended in 200 μl 1% FBS and analyzed by FACS.

To assess the binding properties of the anti-CD28 peptide 2E12V_(H)V_(L), CD28-expressing CHO cells were plated by seeding inindividual wells of a culture plate. The CD20/CD28 purified protein wasthen added to individual wells using a two-fold dilution scheme,extending from 20 μg/ml down to 0.16 μg/ml. The 2E12IgG-VHVL SMIPpurified protein was added to individual seeded wells, again using atwo-fold dilution scheme, i.e., from 20 μg/ml down to 0.16 μg/ml. Onewell received no protein to provide a background control. The plateswere then incubated on ice for one hour, washed once with 200 μl 1% FBSin PBS, and goat anti-human antibody labeled with FITC (Fc Sp, CalTag,Burlingame, Calif.) at 1:100 was added to each well. The plates wereagain incubated on ice for one hour and subsequently washed once with200 μl 1% FBS in PBS. Following re-suspension of the cells in 200 μl 1%FBS, FACS analysis was performed. The results showed that multivalentbinding proteins with the N-terminal CD20 binding domain 1 bound CD20;those proteins having the C-terminal CD28 binding domain 2 in theN-V_(H)-V_(L)-C orientation also bound CD28.

The expressed proteins were shown to bind to CD20 presented on WIL-2Scells (see FIG. 3) and to CD28 presented on CHO cells (refer to FIG. 3)by flow cytometry (FACS), thereby demonstrating that either BD1 or BD2could function to bind the specific target antigen. Each data set on thegraphs in FIG. 3 shows the binding of serial dilutions of the differentmultivalent binding (fusion) proteins over the titration rangesindicated. The data obtained using these initial constructs indicatethat multivalent binding (fusion) proteins with the binding domain 2version using 2e12 in the VHVL orientation express better and bindbetter to CD28 than the form in the VLVH orientation at equivalentconcentrations.

FIG. 4 shows a graphical presentation of the results of binding studiesperformed with purified proteins from each of thesetransfections/constructs. The figure shows binding profiles of theproteins to CD20 expressing WIL-2S cells, demonstrating that themultivalent molecule binds to CD20 as well as the single specificitySMIP at the same concentration. The top and bottom panels for FIG. 5show the binding profiles of the BD2 specificity (2e12=CD28) to CD28 CHOcells. For binding of binding domain 2 to CD28, the orientation of the Vregions affected binding of the 2e12. 2H7-sss-hIgG-STD1-2e12 multivalentbinding proteins with the 2e12 in the VH-VL (HL) orientation showedbinding at a level equivalent to the single specificity SMIP, while the2e12 LH molecule showed less efficient binding at the sameconcentration.

Example 5 Construction of Various Linker Forms of the Multivalent FusionProteins

This example describes the construction of the different linker formslisted in the table shown in FIG. 6.

Construction of C_(H3)-BD2 Linkers H1 Through H7

To explore the effect of C_(H3)-BD2 linker length and composition onexpression and binding of the scorpion molecules, an experiment wasdesigned to compare the existing molecule 2H7sssIgG1-Lx1-2e12HL to alarger set of similar constructs with different linkers. Using2H7sssIgG1-Lx1-2e12HL as template, a series of PCR reactions wereperformed using the primers listed in Oligonucleotide Table 2, whichcreated linkers that varied in length form 0 to 16 amino acids. Theselinkers were constructed as nucleic acid fragments that spanned thecoding region for C_(H3) at the BsrGI site to the end of the nucleicacid encoding the linker-BD2 junction at the EcoRI site.

TABLE 2 Table 2. Sequences of primers used to generateCH3-BD2 linker variants. Name SEQ PCR ID No. Primers Sequence 5′-3′ NO.1 L1-11R GCGATAGAATTCCCAGATCCACCACCGCCCGA 46 GCCACCGCCACCATAATTC 2 L1-6RGCGATAGAATTCCCAGAGCCACCGCCACCATA 47 ATTC 3 L3RGCGTATGAATTCCCCGAGCCACCGCCACCCTTA 48 CCCGGAGACAGG 4 L4RGCGTATGAATTCCCAGATCCACCACCGCCCGAG 49 CCACCGCCACCCTTAC 5 L5RGCGTATGAATTCCCGCTGCCTCCTCCCCCAGATC 50 CACCACCGCC 6 IgBsrG1FGAGAACCACAGGTGTACACCCTG 51 7 L-CPPCPR GCGATAGAATTCGGACAAGGTGGACACCCCTTAC52 CCGGAGACAGGGAGAG

FIG. 6 diagrams the schematic structure of a multivalent binding(fusion) protein and shows the orientation of the V regions for eachbinding domain, the sequence present at linker position 1 (only the Cysresidues are listed), and the sequence and identifier for the linker(s)located at linker position 2 of the molecules.

Example 6 Binding and Functional Studies with Variant Linker Forms ofthe 2H7-IgG-2e12 Prototype Multivalent Fusion Proteins

This example shows the result of a series of expression and bindingstudies on the “prototype” 2H7-sssIgG-Hx-2e12 VHVL construct withvarious linkers (H1-H7) present in the linker position 2. Each of theseproteins was expressed by large-scale COS transient transfection andpurification of the molecules using protein A affinity chromatography,as described in the previous examples. Purified proteins were thensubjected to analyses including SDS-PAGE, Western blotting, bindingstudies analyzed by flow cytometry, and functional assays for biologicalactivity.

Binding Studies Comparing the Different BD2 Orientations

Binding studies were performed as described in the previous examples,except that protein A-purified material was used, and a constant amountof binding (fusion) protein was used for each variant studied, i.e.,0.72 μg/ml. FIG. 7 shows a columnar graph comparing the bindingproperties of each linker variant and 2e12 orientation variant to bothCD20 and CD28 target cells. H1-H6 refer to constructs with the H1-H6linkers and 2e12 in the VH-VL orientation. L1-L6 refer to constructswith the H1-H6 linkers and 2e12 in the VL-VH orientation. The datademonstrate that the binding domain 2 specificity for 2e12 binds muchmore efficiently when present in the HL orientation (samples H1-H6) thanwhen in the LH orientation (samples L1-L6). The effect of linker lengthis complicated by the discovery, as shown in the next set of figures,that molecules with the longer linkers contain some single-specificitycleaved molecules which are missing the CD28 binding specificity at thecarboxy terminus. Other experiments were performed which address thebinding of selected linkers, with the results shown in FIGS. 10, 12, and13.

SDS-PAGE Analysis of Purified H1-H7 Linker Variants

Samples were prepared from purified protein aliquots, containing 8 μgprotein per well, and 2× Tris-Glycine SDS Buffer (Invitrogen) was addedto a 1× final concentration. For reduced samples/gels, 10× reducingbuffer was added to 1× to samples plus Tris-Glycine SDS buffer. Ten (10)μl SeeBlue Marker (Invitrogen, Carlsbad, Calif.) was run to provide MWsize standards. The multivalent binding (fusion) protein variants weresubjected to SDS-PAGE analyses on 4-20% Novex Tris-glycine gels(Invitrogen, San Diego, Calif.). Samples were loaded using NovexTris-glycine SDS sample buffer (2×) under reducing or non-reducingconditions after heating at 95° C. for 3 minutes, followed byelectrophoresis at 175V for 60 minutes. Electrophoresis was performedusing 1× Novex Tris-Glycine SDS Running Buffer (Invitrogen). Gels werestained after electrophoresis in Coomassie SDS PAGE R-250 stain for 30minutes with agitation, and destained for at least one hour. FIG. 8shows the nonreduced and reduced Coomassie stained gels of the[2H7-sss-hIgG P238S/P331S-Hx-2e12 VHVL] multivalent binding (fusion)protein variants, along with TRU-015 and 2e12 HLSMIP as control samples.As the linker length is increased, the amount of protein running atapproximately SMIP size (or 52 kDa) increases. The increase in theamount of protein in this band corresponds with a decrease in the amountof protein in the upper band running at about 90 kDa. The gel dataindicate that the full-length molecule is being cleaved at or near thelinker, to generate a molecule which is missing the BD2 region. Asmaller BD2 fragment is not present, indicating (1) that the nucleotidesequence within the linker sequence may be creating a cryptic splicesite that removes the smaller fragment from the spliced RNA transcript,or (2) that the protein is proteolytically cleaved after translation ofthe full-size polypeptide, and that the smaller BD2 fragment isunstable, i.e., susceptible to proteolytic processing. Western blottingof some of these molecules indicates that the proteins all contain theCD20 BD 1 sequence, but the smaller band is missing the CD28 BD2reactivity. No smaller band migrating at “bare” scFv size (25-27 kDa)was observed on any gels or blots, indicating that this smaller peptidefragment is not present in the samples.

Western Blot Binding of BD1 and BD2 by 2H7 Specific Fab or CD28mIg

FIG. 9 shows the results of Western blotting of the 2H7-sss-hIgG-H6multivalent binding (fusion) proteins compared to eachsingle-specificity SMIP.

Electrophoresis was performed under non-reducing conditions, and withoutboiling samples prior to loading. After electrophoresis, proteins weretransferred to PVDF membranes using a semi-dry electroblotter apparatus(Ellard, Seattle, Wash.) for 1 hour at 100 mAmp. Membranes were blockedin BLOTTO (5% nonfat milk in PBS) overnight with agitation. FIG. 9A:Membranes were incubated with the AbyD02429.2, a Fab directed to the 2H7antibody, at 5 μg/ml in BLOTTO for one hour, then washed 3 times for 5minutes each in PBS-0.5% Tween 20. Membranes were then incubated in6×His-HRP for one hour at a concentration of 0.5 μg/ml. Blots werewashed three times for 15 minutes each in PBST. Wet membranes wereincubated with ECL solution for 1 minute, followed by exposure to X-omatfilm for 20 seconds.

FIG. 9B: Membranes were incubated with CD28Ig (Ancell, Bayport, Minn.)at 10 μg/ml in BLOTTO, then washed three times for 15 minutes each inPBS-0.5% Tween 20. Membranes were then incubated in goat anti-mouse HRPconjugate (CalTag, Burlingame, Calif.) at 1:3000 in BLOTTO. Membraneswere washed three times, for 15 minutes each, then incubated in ECLsolution for 1 minute, followed by exposure to X-omat film for 20seconds. The results from the Western blots indicated that the CD28binding domain was present in the multivalent “monomer” fractionmigrating at approximately 90 kDa, and in higher order forms. No bandwas detectable migrating at the position expected for a single SMIP orbare scFv size fragment. When the CD20 anti-idiotype Fab was used, aSMIP-sized fragment was detected, indicating the presence of a peptidefragment containing (2H7-sss-hIgG), and missing the CD28 scFv BD2portion of the fusion protein.

Binding Studies on Selected Linkers

FIG. 10 shows the results of binding studies performed on the purified2H7-sss-hIgG-Hx-2e12 fusion proteins. Binding studies were performed toassess the bispecific binding properties of the CD20/CD28 multispecificbinding peptides. Initially, WIL2-S cells were plated using conventionaltechniques. To the seeded plates, CD20/CD28 purified protein was added,using two-fold titrations across the plate from 20 μg/ml down to 0.16μg/ml. A two-fold dilution series of TRU-015 (source of bindingdomain 1) purified protein was also added to seeded plate wells, theconcentration of TRU-015 extending from 20 μg/ml down to 0.16 μg/ml. Onewell containing no protein served as a background control.

Seeded plates containing the proteins were incubated on ice for onehour. Subsequently, the wells were washed once with 200 μl 1% FBS inPBS. Goat anti-human antibody labeled with FITC (Fc Sp) at 1:100 wasthen added to each well, and the plates were again incubated on ice forone hour. The plates were then washed once with 200 μl 1% FBS in PBS andthe cells were re-suspended in 200 μl 1% FBS and analyzed by FACS.

To assess the binding properties of the anti-CD28 peptide 2E12V_(H)V_(L), CD28-expressing CHO cells were plated by seeding inindividual wells of a culture plate. The CD20/CD28 purified protein wasthen added to individual wells using a two-fold dilution scheme,extending from 20 μg/ml down to 0.16 μg/ml. The 2E12IgGvHvL SMIPpurified protein was added to individual seeded wells, again using atwo-fold dilution scheme, i.e., from 20 μg/ml down to 0.16 μg/ml. Onewell received no protein to provide a background control. The plateswere then incubated on ice for one hour, washed once with 200 μl 1% FBSin PBS, and goat anti-human antibody labeled with FITC (Fc Sp) at 1:100was added to each well. The plates were again incubated on ice for onehour and subsequently washed once with 200 μl 1% FBS in PBS. Followingre-suspension of the cells in 200 μl 1% FBS, FACS analysis wasperformed. The expressed proteins were shown to bind to CD20 presentedon WIL-2S cells (see FIG. 10A) and to CD28 presented on CHO cells (referto FIG. 10B) by flow cytometry (FACS), thereby demonstrating that eitherBD 1 or BD2 could function to bind the specific target antigen. Inaddition, the linker used (H1-H6) was not found to significantly affectbinding avidity to target antigen.

SEC Fractionation of Multivalent Binding (Fusion) Proteins. The binding(fusion) protein was purified from cell culture supernatants by proteinA Sepharose affinity chromatography over a GE Healthcare XK 16/40column. After binding of protein to the column, the column was washed indPBS, then 1.0 M NaCl, 20 mM sodium phosphate pH 6.0, and then 25 mMNaCl, 25 mN NaOAc, pH 5.0, to remove nonspecific binding proteins. Boundprotein was eluted from the column in 100 mM Glycine (Sigma), pH 3.5,and brought to pH 5.0 with 0.5 M 2-(N-Morpholino) ethanesulfonic acid(MES), pH 6.0. Protein samples were concentrated to 25 mg/ml usingconventional techniques in preparation for GPC purification. Sizeexclusion chromatography (SEC) was performed on a GE Healthcare AKTAExplorer 100 Air apparatus, using a GE healthcare XK column and Superdex200, preparative grade (GE healthcare).

FIG. 12 shows a table summarizing the results of SEC fractionation ofthe different binding (fusion) proteins. With increasing linker length,the complexity of the molecules in solution also increases, making itdifficult to isolate peak of interest, or POI from higher order forms byHPLC. The H7 linker seems to resolve much of this complexity into a morehomogeneous form in solution, so that the soluble forms migrate mostlyas a single POI at approximately 172 kDa.

Additional Binding Studies

A second series of experiments was performed (see FIGS. 12 and 13) witha smaller subset of multivalent binding (fusion) proteins, this timecomparing linkers H3, H6, and H7. The data demonstrate that the bindinglevel drops more significantly for CD28 than for CD20 binding, but bothdrop slightly as linker length increases. Further, the data showed thatthe H7 linker exhibited the highest level of binding to both antigens.These data were obtained using protein A-purified multivalent binding(fusion) proteins, but were not further purified by SEC, so multipleforms of the molecules may have been present in solution. The resultsalso indicated that the truncated form may have been less stable thanthe true multivalent polypeptide, since the binding curves do not appearto fully reflect the significant amount of single specificity formpresent in solution for linker H6.

Demonstration of Multispecific Binding From a Single Molecule

An alternative binding assay was performed (see FIG. 13), where bindingto CD20 on the surface of WIL-2S cells was detected with a reagentspecific for the CD28 BD2, thereby demonstrating that simultaneousbinding may occur to both target antigens, engaging both BD1 and BD2 onthe same multispecific binding (fusion) protein (refer to FIG. 12) Thisassay demonstrates the multispecific binding property of the proteins.

Example 7 Construction of Multispecific Binding (Fusion) Proteins withAlternative Specificities in BD2

In addition to the prototype CD20-CD28 multispecific binding molecule,two other forms were made with alternative binding domain 2 regions,including CD37 and CD3 binding domains. The molecules were also madewith several of the linker domains described for the[2H7-sss-IgG-Hx/STDx-2e12 HL] multispecific binding (fusion) proteins.The construction of these additional multispecific binding (fusion)molecules are described below.

Anti-CD37 Binding Domain Construction

TABLE 3 Table 3. Oligonucleotide primers used to generateG28-1 anti-CD37 binding domains for both SMIP molecules and scorpions.SEQ No. Name Sequence ID NO. 23 G281LH-NheR ACTGCTGCAGCTGGACCGCGCT 53AGCTCCGCCGCCACCCGAC 24 G281LH-NheF GGCGGAGCTAGCGCGGTCCAGC 54TGCAGCAGTCTGGACCTG 25 G281-LH-LPinF GCGATCACCGGTGACATCCAGAT 55GACTCAGTCTCCAG 26 G281-LH-HXhoR GCGATACTCGAGGAGACGGTGAC 56TGAGGTTCCTTGAC 27 G281-LH-LEcoF GCGATCGAATTCAGACATCCAGAT 57GACTCAGTCTCCAG 28 G281-LH-HXbaR GCGATTCTAGATTAGGAAGAGACG 58GTGACTGAGGTTCCTTGAC 29 G281-HL-HF GCGATAACCGGTGCGGTCCAGCTG 59CAGCAGTCTGGAC 30 G281-HL-HR3 GACCCACCACCGCCCGAGCCACCG 60CCACCAGAAGAGACGGTGACTGAGG TTC 31 G281-HL-HR2 ACTCCCGCCTCCTCCTGATCCGCCG61 CCACCCGACCCACCACCGCCCGAG 32 G281-HL-HNheR GAGTCATCTGGATGTCGCTAGCACTC62 CCGCCTCCTCCTGATC 33 G281-HL-LNheF ATCAGGAGGAGGCGGGAGTGCTAGC 63GACATCCAGATGACTCAGTC 34 G281-HL-LXhoR GCGATACTCGAGCCTTTGATCTCCAG 64TTCGGTGCCTC 35 G281-HL-LXbaR GCGATATCTAGACTCAACCTTTGATCT 65CCAGTTCGGTGCCTC 36 G281-HL-EcoF GCGATAGAATTCGCGGTCCAGCTGCA 66GCAGTCTGGAC

The G28-1 scFv (SEQ ID NO:102) was converted to the G28-1 LH SMIP by PCRusing the primers in Table X above. Combining primers 23 and 25 with 10ng G28-1 scFv, the VK was amplified for 30 cycles of 94 C, 20 seconds,58 C, 15 seconds, 68 C, 15 seconds using Platinum PCR SupermixHi-Fidelity PCR mix (Invitrogen, Carlsbad, Calif.) in an ABI 9700Thermalcycler. The product of this PCR had the restriction sites PinAI(AgeI) at the 5′ end of the VK and NheI at the end of the scFv (G4S)3linker. The VH was similarly altered by combining primers 24 and 26 with10 ng G28-1 scFv in a PCR run under the identical conditions as with theVK above. This PCR product had the restriction sites NheI at the 5′ endof the VH and XhoI at the 3′ end. Because significant sequence identityoverlap was engineered into primers 23 and 24, the VK and VH werediluted 5-fold, then added at a 1:1 ratio to a PCR using the flankingprimers 25 and 26 and a full-length scFv was amplified as above bylengthening the 68 C extension time from 15 seconds to 45 seconds. ThisPCR product represented the entire G28-1 scFv as a PinAI-XhoI fragmentand was purified by MinElute column (Qiagen,) purification to removeexcess primers, enzymes and salts. The eluate was digested to completionwith PinAI (Invitrogen) and XhoI (Roche) in 1×H buffer (Roche,) at 37 Cfor 4 hours in a volume of 50 μL. The digested PCR product was thenelectrophoresed in a 1% agarose gel, the fragment was removed from thegel and re-purified on a MinElute column using buffer QG and incubatingthe gel-buffer mix at 50 C for 10 minutes with intermittent mixing todissolve the agarose after which the purification on the column wasidentical for primer removal post-PCR. 3 μL PinAI-XhoI digested G28-1 LHwas combined with 1 μL PinAI-XhoI digested pD18-n2H7sssIgG1 SMIP in a 10μL reaction with 5 μL 2× LigaFast Ligation Buffer (Promega, Madison,Wis.) and 1 μL T4 DNA ligase (Roche), mixed well and incubated at roomtemperature for 10 minutes. 3 μL of this ligation was then transformedinto competent TOP 10 (Invitrogen) using the manufacturer's protocol.These transformants were plated on LB agar plates with 100 μg/mlcarbenicillin (Teknova,) and incubated overnight at 37 C. After 18 hoursof growth, colonies were picked and inoculated into 1 ml T-Broth(Teknova,) containing 100 μg/ml carbenicillin in a deep well 96-wellplate and grown overnight in a 37 C shaking incubator. After 18-24 hoursof growth, DNA was isolated from each overnight culture using theQIAprep 96 Turbo Kit (Qiagen) on the BioRobot8000 (Qiagen). 10 μL fromeach clone was then digested with both HindIII and XhoI restrictionenzymes in 1×B buffer in a 15 μL reaction volume. The digested DNA waselectrophoresed on 1% agarose E-gels (Invitrogen, CA) for restrictionsite analysis. Clones that contained a HindIII-XhoI fragment of thecorrect size were sequence verified. The G28-1 HL SMIP was constructedin a similar manner by placing a PinAI site on the 5′ end and a (G4S)4linker ending in an Nhe I site of the G28-1 VH using primers 29, 30 31and 32 from Table X above. The VK was altered by PCR using primers 33and 34 from Table X such that an NheI site was introduced at the 5′ endof the VK and XhoI at the 3′ end. These PCRs were then combined as aboveand amplified with the flanking primers 29 and 34 to yield an intactG28-1 scFv DNA in the VH-VL orientation which was cloned into PinAI-XhoIdigested pD18-(n2H7)sssIgG1 SMIP exactly as with the G28-1 LH SMIP.

2H7sssIgG1-STD1-G28-1 LH/HL Construction

Using the G28-1 LH and G28-1 HL SMIPs as templates, the LH and HLanti-CD37 binding domains were altered by PCR such that their flankingrestriction sites were compatible with the scorpion cassette. An EcoRIsite was introduced at the 5′ end of each scFv using either primer 27(LH) or 36 (HL) and a stop codon/XbaI site at the 3′ end using eitherprimer 28 (LH) or 35 (HL). The resulting DNAs were cloned intoEcoRI-XbaI digested pD18-2H7sssIgG-STD1.

2H7sssIgG1-Hx-G28-1 HL Construction

2H7sssIgG1-Hx-2e12 HL DNAs were digested with BsrGI and EcoRI and the325 bp fragment consisting of the C-terminal end of the IgG1 and linker.These were substituted for the equivalent region in2H7sssIgG1-STD1-G19-4 HL by removal of the STD1 linker using BsrGI-EcoRIand replacing it with the corresponding linkers from the2H7sssIgG1-Hx-2e12 HL clones.

Anti-CD3 Binding Domain Construction

TABLE 4 Table 4. Oligonucleotides used to generate anti-CD3 binding domain from the G19-4 scFv sequence. SEQ ID No. NameSequence NO. 37 194-LH-LF1 GCGTATGAACCGGTGACATCCAGAT 67 GACACAGACTACATC38 194-LF2 ATCCAGATGACACAGACTACATCCTC 68 CCTGTCTGCCTCTCTGGGAGACAG 39194-LF3 GTCTGCCTCTCTGGGAGACAGAGTCA 69 CCATCAGTTGCAGGGCAAGTCAGGAC 40194-LF4 GTTGCAGGGCAAGTCAGGACATTCGC 70 AATTATTTAAACTGGTATCAGCAG 41194-LF5 ATTTAAACTGGTATCAGCAGAAACCAG 71 ATGGAACTGTTAAACTCCTGATC 42194-LF6 GAACTGTTAAACTCCTGATCTACTACA 72 CATCAAGATTACACTCAGGAGTC 43194-LF7 CAAGATTACACTCAGGAGTCCCATCAA 73 GGTTCAGTGGCAGTGGGTCTGGAAC 44194-LR7 CAGGTTGGCAATGGTGAGAGAATAATC 74 TGTTCCAGACCCACTGCCACTGAAC 45194-LR6 GCAAAAGTAAGTGGCAATATCTTCTGGT 75 TGCAGGTTGGCAATGGTGAGAG 46194-LR5 GAACGTCCACGGAAGCGTATTACCC 76 TGTTGGCAAAAGTAAGTGGCAATATC 47194-LR4 CGTTTGGTTACCAGTTTGGTGCCTCCAC 77 CGAACGTCCACGGAAGCGTATTAC 48194-LR3 ACCACCGCCCGAGCCACCGCCACC 78 CCGTTTGGTTACCAGTTTGGTG 49 194-LR2GCTAGCGCTCCCACCTCCTCCAGATCCA 79 CCACCGCCCGAGCCACCGCCAC 50 194-LH-LR1GTTGCAGCTGGACCTCGCTAGCGCT 80 CCCACCTCCTCCAGATC 51 194-LH-HF1GATCTGGAGGAGGTGGGAGCGCTAGC 81 GAGGTCCAGCTGCAACAGTCTGGACCTG 52 194-HF2AGCTGCAACAGTCTGGACCTGAACT 82 GGTGAAGCCTGGAGCTTCAATGAAG 53 194-HF3AGCCTGGAGCTTCAATGAAGATTTCC 83 TGCAAGGCCTCTGGTTACTCATTC 54 194-HF4GCAAGGCCTCTGGTTACTCATTCACT 84 GGCTACATCGTGAACTGGCTGAAGCAG 55 194-HF5ATCGTGAACTGGCTGAAGCAGAGCC 85 ATGGAAAGAACCTTGAGTGGATTGGAC 56 194-HF6GAACCTTGAGTGGATTGGACTTATTA 86 ATCCATACAAAGGTCTTACTACCTAC 57 194-HR6AATGTGGCCTTGCCCTTGAATTTCTG 87 GTTGTAGGTAGTAAGACCTTTGTATG 58 194-HR5CATGTAGGCTGTGCTGGATGACTTGT 88 CTACAGTTAATGTGGCCTTGCCCTTG 59 194-HR4ACTGCAGAGTCTTCAGATGTCAGACTG 89 AGGAGCTCCATGTAGGCTGTGCTGGATG 60 194-HR3ACCATAGTACCCAGATCTTGCACAG 90 TAATAGACTGCAGAGTCTTCAGATGTC 61 194-HR2GCGCCCCAGACATCGAAGTACCAGTC 91 CGAGTCACCATAGTACCCAGATCTTG 62 194-LH-HR1GCGAATACTCGAGGAGACGGTGACCG 92 TGGTCCCTGCGCCCCAGACATCGAAG 63 194-HL-HF1GCGTATGAACCGGTGAGGTCCAGC 93 TGCAACAGTCTGGACCTG 64 194-HL-HR1ACCGCCACCAGAGGAGACGGTGACCGT 94 GGTCCCTGCGCCCCAGACATCGAAGTAC 65194-HL-HR0 ACCTCCTCCAGATCCACCACCGCCCG 95 AGCCACCGCCACCAGAGGAGACGGTG 66194-HL-LF1 GCGGGGGAGGTGGCAGTGCTAGCGA 96 CATCCAGATGACACAGACTACATC 67194-HL-LR3Xho GCGAATACTCGAGCGTTTGGTTACCA 97 GTTTGGTG 68 194-HL-LR3XbaGCGATATCTAGATTACCGTTTGGTTAC 98 CAGTTTGGTG 69 194-HL-HF1R1GCGTATGAGAATTCAGAGGTCCAGCTG 99 CAACAGTCTGGACCTG 70 194-LH-LF1R1GCGTATGAGAATTCTGACATCCAGA 100 TGACACAGACTACATC 71 194-LH-HR1XbaGCGTATCTAGATTAGGAGACGGTGACC 101 GTGGTCCCTGCGCCCCAGACATCGAAG

The G19-4 binding domain was synthesized by extension of overlappingoligonucleotide primers as described previously. The light chain PCR wasdone in two steps, beginning by combining primers 43/44, 42/45, 41/46and 40/47 at concentrations of 5 uM, 10 μM, 20 μM and 40 μM,respectively, in Platinum PCR Supermix Hi-Fidelity for 30 cycles of 94°C., 20 seconds, 60° C., 10 seconds, 68° C., 15 seconds. 1 μL of theresultant PCR product was reamplified using a primer mix of 39/48 (10μM), 38/49 (20 μM) and 37/50 (40 μM) for the LH or 66/67 (40 μM) for theHL orientation, using the same PCR conditions with the exception of the68 C extension which was increased to 25 seconds. The VK in the LHorientation was bounded by PinAI at the 5′ end and NheI at the 3′ end,while the HL orientation had NheI at the 5′ end and XhoI at the 3′ end.

To synthesize the heavy chain, primer mixes with the same concentrationsas above were prepared by combining primers 56/57, 55/58, 54/59 and53/60 for the first PCR step. In the second PCR, primers 52/61 (20 μM)and 51/62 (50 μM) were amplified with 1 μl from the first PCR using thesame PCR conditions as with the second PCR of the light chain to makethe LH orientation with NheI at the 5′ end and XhoI at the 3′ end.Primers 52/61 (10 μM), 63/64 (20 μM), 63 (20 μM)/65 (40 μM) and 63 (20μM)/5 (80 μM) were combined in a second PCR with 1 uL from the previousPCR to create the heavy chain in the HL orientation with PinAI at the 5′end and NheI at the 3′ end. As with previous constructs, sufficientoverlap was designed into the primers centered around the NheI site suchthat the G19-4 LH was synthesized by combining the heavy and light chainPCRs in the LH orientation and reamplifying with the flanking primers,37 and 62 and the G19-4 HL was synthesized by combining the HL PCRs andre-amplifying with primers 63 and 67.

Full-length G19-4 LH/HL PCR products were separated by agarose gelelectrophoresis, excised from the gel and purified with Qiagen MinElutecolumns as described earlier. These DNAs were then TOPO-cloned intopCR2.1 (Invitrogen), transformed into TOP10 and colonies screened firstby EcoRI fragment size, then by DNA sequencing. G19-4 LH/HL were thencloned into pD18-IgG1 via PinAI-XhoI for expression in mammalian cells.

2H7sssIgG1-STD1-G19-4 LH/HL Construction

Using the G19-4 LH and G19-4 HL SMIPs as templates, the LH and HLanti-CD3 binding domains were altered by PCR such that their flankingrestriction sites were compatible with the scorpion cassette. An EcoRIsite was introduced at the 5′ end of each scFv using either primer 27(LH) or 36 (HL) and a stop codon/XbaI site at the 3′ end using eitherprimer 28 (LH) or 35 (HL). The resulting DNAs were cloned intoEcoRI-XbaI digested pD18-2H7sssIgG-STD1.

2H7sssIgG1-Hx-G19-4 HL Construction

2H7sssIgG1-Hx-2e12 HL DNAs were digested with BsrGI and EcoRI and the325 bp fragment consisting of the C-terminal end of the IgG1 and linker.These were substituted for the equivalent region in2H7sssIgG1-STD1-G19-4 HL by removal of the STD 1 linker usingBsrGI-EcoRI and replacing it with the corresponding linkers from the2H7sssIgG1-Hx-2e12 HL clones.

Apparent from a consideration of the variety of multivalent bindingproteins disclosed herein are features of the molecules that areamenable to combination in forming the molecules of the invention. Thosefeatures include binding domain 1, a constant sub-region, including ahinge or hinge-like domain, a linker domain, and a binding domain 2. Theintrinsic modularity in the design of these novel binding proteins makesit straightforward for one skilled in the art to manipulate the DNAsequence at the N-terminal and/or C-terminal ends of any desirablemodule such that it can be inserted at almost any position to create anew molecule exhibiting altered or enhanced functionality compared tothe parental molecule(s) from which it was derived. For example, anybinding domain derived from a member of the immunoglobulin superfamilyis contemplated as either binding domain 1 or binding domain 2 of themolecules according to the invention. The derived binding domainsinclude domains having amino acid sequences, and even encodingpolynucleotide sequences, that have a one-to-one correspondence with thesequence of a member of the immunoglobulin superfamily, as well asvariants and derivatives that preferably share 80%, 90%, 95%, 99%, or99.5% sequence identity with a member of the immunoglobulin superfamily.These binding domains (1 and 2) are preferably linked to other modulesof the molecules according to the invention through linkers that mayvary in sequence and length as described elsewhere herein, provided thatthe linkers are sufficient to provide any spacing and flexibilitynecessary for the molecule to achieve a functional tertiary structure.Another module of the multivalent binding proteins is the hinge region,which may correspond to the hinge region of a member of theimmunoglobulin superfamily, but may be a variant thereof, such as the“CSC” or “SSS” hinge regions described herein. Also, the constantsub-region comprises a module of the proteins according to the inventionthat may correspond to a sub-region of a constant region of animmunoglobulin superfamily member, as is typified by the structure of ahinge-C_(H2)-C_(H3) constant sub-region. Variants and derivatives ofconstant sub-regions are also contemplated, preferably having amino acidsequences that share 80%, 90%, 95%, 99%, or 99.5% sequence identity witha member of the immunoglobulin superfamily.

Exemplary primary structures of the features of such molecules arepresented in Table 5, which discloses the polynucleotide and cognateamino acid sequence of illustrative binding domains 1 and 2, as well asthe primary structure of a constant sub-region, including a hinge orhinge-like domain, and a linker that may be interposed, e.g., betweenthe C-terminal end of a constant sub-region and the N-terminal end of abinding domain 2 region of a multivalent binding protein. Additionalexemplars of the molecules according to the invention include theabove-described features wherein, e.g., either or both of bindingdomains 1 and 2 comprise a domain derived from a V_(L) or V_(L)-likedomain of a member of the immunoglobulin superfamily and a V_(H) orV_(H)-like domain derived from the same or a different member of theimmunoglobulin superfamily, with these domains separated by a linkertypified by any of the linkers disclosed herein. Contemplated aremolecules in which the orientation of these domains is V_(L)-V_(H) orV_(H)-V_(L) for BD1 and/or BD2. A more complete presentation of theprimary structures of the various features of the multivalent bindingmolecules according to the invention is found in the table appended atthe end of this disclosure. The invention further comprehendspolynucleotides encoding such molecules.

TABLE 5 Table 5. Primary structures (polynucoleotide and cognate aminoacid sequences) of exemplary features of multivalent binding molecules.SEQ ID NOS. (amino Binding acid Domain Nucleotide SequenceAmino Acid Sequence sequence 2H7 LH atggattttcaagtgcagattttcagmdfqvqifsfllisasvimsrgqivls 1 (2) cttcctgctaatcagtgcttcagtcaqspailsaspgekvtmtcrasssvsym taatgtccagaggacaaattgttctchwyqqkpgsspkpwiyapsnlasgvpa tcccagtctccagcaatcctgtctgcrfsgsgsgtsysltisrveaedaatyy atctccaggggagaaggtcacaatgacqqwsfnpptfgagtklelkdgggsgg cttgcagggccagctcaagtgtaagtggsggggssqaylqqsgaesvrpgasv tacatgcactggtaccagcagaagcckmsckasgytftsynmhwvkqtprqgl aggatcctcccccaaaccctggatttewigaiypgngdtsynqkfkgkatltv atgccccatccaacctggcttctggadkssstaymqlssltsedsavyfcarv gtccctgctcgcttcagtggcagtggvyysnsywyfdvwgtgttvtvs gtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgct gccacttattactgccagcagtggagttttaacccacccacgttcggtgctg ggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctgg aggaggtgggagctctcaggcttatctacagcagtctggggctgagtcggtg aggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacattta ccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatg gattggagctatttatccaggaaatggtgatacttcctacaatcagaagttc aagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgc agctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagt ggtgtactatagtaactcttactggtacttcgatgtctggggcacagggacc acggtcaccgtctct 2e12 LHatggattttcaagtgcagattttcag MDFQVQIFSFLLISASVIMSRGVDIVL 3 (4)cttcctgctaatcagtgcttcagtca TQSPASLAVSLGQRATISCRASESVEYtaatgtccagaggagtcgacattgtg YVTSLMQWYQQKPGQPPKLLISAASNVctcacccaatctccagcttctttggc ESGVPARFSGSGSGTDFSLNIHPVEEDtgtgtctctaggtcagagagccacca DIAMYFCQQSRKVPWTFGGGTKLEIKRtctcctgcagagccagtgaaagtgtt GGGGSGGGGSGGGGSQVQLKESGPGLVgaatattatgtcacaagtttaatgca APSQSLSITCTVSGFSLTGYGVNWVRQgtggtaccaacagaaaccaggacagc PPGKGLEWLGMIWGDGSTDYNSALKSRcacccaaactcctcatctctgctgct LSITKDNSKSQVFLKMNSLQTDDTARYagcaacgtagaatctggggtccctgc YCARDGYSNFHYYVMDYWGQGTSVTVScaggtttagtggcagtgggtctggga S cagactttagcctcaacatccatcctgtggaggaggatgatattgcaatgta tttctgtcagcaaagtaggaaggttccatggacgttcggtggaggcaccaag ctggaaatcaaacggggtggcggtggatccggcggaggtgggtcgggtggcg gcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctc acagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctat ggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctgggaa tgatatggggtgatggaagcacagactataattcagctctcaaatccagact atcgatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagt ctgcaaactgatgacacagccagatactactgtgcccgagatggttatagta actttcattactatgttatggactactggggtcaaggaacctcagtcaccgt ctcctct 2e12 HL atggattttcaagtgcagattttcagMDFQVQIFSFLLISASVIMSRGVQVQL 5 (6) cttcctgctaatcagtgcttcagtcaKESGPGLVAPSQSLSITCTVSGFSLTG taatgtccagaggagtccaggtgcagYGVNWVRQPPGKGLEWLGMIWGDGSTD ctgaaggagtcaggacctggcctggtYNSALKSRLSITKDNSKSQVFLKMNSL ggcgccctcacagagcctgtccatcaQTDDTARYYCARDGYSNFHYYVMDYWG catgcaccgtctcagggttctcattaQGTSVTVSSGGGGSGGGGSGGGGSGGG accggctatggtgtaaactgggttcgGSDIVLTQSPASLAVSLGQRATISCRA ccagcctccaggaaagggtctggagtSESVEYYVTSLMQWYQQKPGQPPKLLI ggctgggaatgatatggggtgatggaSAASNVESGVPARFSGSGSGTDFSLNI agcacagactataattcagctctcaaHPVEEDDIAMYFCQQSRKVPWTFGGGT atccagactatcgatcaccaaggaca KLEIKRactccaagagccaagttttcttaaaa atgaacagtctgcaaactgatgacacagccagatactactgtgcccgagatg gttatagtaactttcattactatgttatggactactggggtcaaggaacctc agtcaccgtctcctctgggggtggaggctctggtggcggtggatccggcgga ggtgggtcgggtggcggcggatctgacattgtgctcacccaatctccagctt ctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtga aagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaacca ggacagccacccaaactcctcatctctgctgctagcaacgtagaatctgggg tccctgccaggtttagtggcagtgggtctgggacagactttagcctcaacat ccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtagg aaggttccatggacgttcggtggaggcaccaagctggaaatcaaacgt G28-1 accggtgacatccagatgactcagtcDIQMTQSPASLSASVGETVTITCRTSE 102 (103) LH tccagcctccctatctgcatctgtggNVYSYLAWYQQKQGKSPQLLVSFAKTL gagagactgtcaccatcacatgtcgaAEGVPSRFSGSGSGTQFSLKISSLQPE acaagtgaaaatgtttacagttatttDSGSYFCQHHSDNPWTFGGGTELEIKG ggcttggtatcagcagaaacagggaaGGGSGGGGSGGGGSASAVQLQQSGPEL aatctcctcagctcctggtctcttttEKPGASVKISCKASGYSFTGYNMNWVK gcaaaaaccttagcagaaggtgtgccQNNGKSLEWIGNIDPYYGGTTYNRKFK atcaaggttcagtggcagtggatcagGKATLTVDKSSSTAYMQLKSLTSEDSA gcacacagttttctctgaagatcagcVYYCARSVGPMDYWGQGTSVTVS agcctgcagcctgaagattctggaagttatttctgtcaacatcattccgata atccgtggacgttcggtggaggcaccgaactggagatcaaaggtggcggtgg ctcgggcggtggtgggtcgggtggcggcggatctgctagcgcagtccagctg cagcagtctggacctgagctggaaaagcctggcgcttcagtgaagatttcct gcaaggcttctggttactcattcactggctacaatatgaactgggtgaagca gaataatggaaagagccttgagtggattggaaatattgatccttattatggt ggtactacctacaaccggaagttcaagggcaaggccacattgactgtagaca aatcctccagcacagcctacatgcagctcaagagtctgacatctgaggactc tgcagtctattactgtgcaagatcggtcggccctatggactactggggtcaa ggaacctcagtcaccgtctcgag G28-1accggtgaggtccagctgcaacagtc EVQLQQSGPELVKPGASMKISCKASGY 104 (105) HLtggacctgaactggtgaagcctggag SFTGYIVNWLKQSHGKNLEWIGLINPYcttcaatgaagatttcctgcaaggcc KGLTTYNQKFKGKATLTVDKSSSTAYMtctggttactcattcactggctacat ELLSLTSEDSAVYYCARSGYYGDSDWYcgtgaactggctgaagcagagccatg FDVWGAGTTVTVSSGGGGSGGGGSGGGgaaagaaccttgagtggattggactt GSGGGGSASDIQMTQTTSSLSASLGDRattaatccatacaaaggtcttactac VTISCRASQDIRNYLNWYQQKPDGTVKctacaaccagaaattcaagggcaagg LLIYYTSRLHSGVPSRFSGSGSGTDYSccacattaactgtagacaagtcatcc LTIANLQPEDIATYFCQQGNTLPWTFGagcacagcctacatggagctcctcag GGTKLVTKRS tctgacatctgaagactctgcagtctattactgtgcaagatctgggtactat ggtgactcggactggtacttcgatgtctggggcgcagggaccacggtcaccg tctcctctggtggcggtggctcgggcggtggtggatctggaggaggtgggag cgggggaggtggcagtgctagcgacatccagatgacacagactacatcctcc ctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcagg acattcgcaattatttaaactggtatcagcagaaaccagatggaactgttaa actcctgatctactacacatcaagattacactcaggagtcccatcaaggttc agtggcagtgggtctggaacagattattctctcaccattgccaacctgcaac cagaagatattgccacttacttttgccaacagggtaatacgcttccgtggac gttcggtggaggcaccaaactggtaa ccaaacgctcgagG19-4 accggtgacatccagatgacacagac DIQMTQTTSSLSASLGDRVTISCRASQ 106 (107)LH tacatcctccctgtctgcctctctgg DIRNYLNWYQQKPDGTVKLLIYYTSRLgagacagagtcaccatcagttgcagg HSGVPSRFSGSGSGTDYSLTIANLQPEgcaagtcaggacattcgcaattattt DIATYFCQQGNTLPWTFGGGTKLVTKRaaactggtatcagcagaaaccagatg GGGGSGGGGSGGGGSASEVQLQQSGPEgaactgttaaactcctgatctactac LVKPGASMKISCKASGYSFTGYIVNWLacatcaagattacactcaggagtccc KQSHGKNLEWIGLINPYKGLTTYNQKFatcaaggttcagtggcagtgggtctg KGKATLTVDKSSSTAYMELLSLTSEDSgaacagattattctctcaccattgcc AVYYCARSGYYGDSDWYFDVWGAGTTVaacctgcaaccagaagatattgccac TVSS ttacttttgccaacagggtaatacgcttccgtggacgttcggtggaggcacc aaactggtaaccaaacggggtggcggtggctcgggcggtggtggatctggag gaggtgggagcgctagcgaggtccagctgcaacagtctggacctgaactggt gaagcctggagcttcaatgaagatttcctgcaaggcctctggttactcattc actggctacatcgtgaactggctgaagcagagccatggaaagaaccttgagt ggattggacttattaatccatacaaaggtcttactacctacaaccagaaatt caagggcaaggccacattaactgtagacaagtcatccagcacagcctacatg gagctcctcagtctgacatctgaagactctgcagtctattactgtgcaagat ctgggtactatggtgactcggactggtacttcgatgtctggggcgcagggac cacggtcaccgtctcctcgag G19-4accggtgaggtccagctgcaacagtc EVQLQQSGPELVKPGASMKISCKASGY 108 (109) HLtggacctgaactggtgaagcctggag SFTGYIVNWLKQSHGKNLEWIGLINPYcttcaatgaagatttcctgcaaggcc KGLTTYNQKFKGKATLTVDKSSSTAYMtctggttactcattcactggctacat ELLSLTSEDSAVYYCARSGYYGDSDWYcgtgaactggctgaagcagagccatg FDVWGAGTTVTVSSGGGGSGGGGSGGGgaaagaaccttgagtggattggactt GSASDIQMTQTTSSLSASLGDRVTISCattaatccatacaaaggtcttactac RASQDIRNYLNWYQQKPDGTVKLLIYYctacaaccagaaattcaagggcaagg TSRLHSGVPSRFSGSGSGTDYSLTIANccacattaactgtagacaagtcatcc LQPEDIATYFCQQGNTLPWTFGGGTKLagcacagcctacatggagctcctcag VTKRS tctgacatctgaagactctgcagtctattactgtgcaagatctgggtactat ggtgactcggactggtacttcgatgtctggggcgcagggaccacggtcaccg tctcctctggtggcggtggctcgggcggtggtggatctggaggaggtgggag cgctagcgacatccagatgacacagactacatcctccctgtctgcctctctg ggagacagagtcaccatcagttgcagggcaagtcaggacattcgcaattatt taaactggtatcagcagaaaccagatggaactgttaaactcctgatctacta cacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtct ggaacagattattctctcaccattgccaacctgcaaccagaagatattgcca cttacttttgccaacagggtaatacgcttccgtggacgttcggtggaggcac caaactggtaaccaaacgctcgag SEQ ID NO. Hinge(amino acid Region Nucleotide Sequence Amino Acid Sequence sequence)sss(s)- gagcccaaatcttctgacaaaact EPKSSDKTHTSPPSS 230 (231) hIgG1cacacatctccaccgagctca csc(s)- gagcccaaatcttgtgacaaaact EPKSCDKTHTSPPCS232 (233) hIgG1 cacacatctccaccgtgctca ssc(s)- gagcccaaatcttctgacaaaactEPKSSDKTHTSPPCS 110 (111) hIgG1 cacacatctccaccgtgctca scc(s)-gagcccaaatcttctgacaaaact EPKSSDKTHTCPPCS 112 (113) hIgG1cacacatgtccaccgtgctca css(s)- gagcccaaatcttgtgacaaaact EPKSCDKTHTSPPSS114 (115) hIgG1 cacacatctccaccgagctca scs(s)- gagcccaaatcttgtgacaaaactEPKSSDKTHTCPPSS 116 (117) hIgG1 cacacatgtccaccgagctca ccc(s)-gagcccaaatcttgtgacaaaact EPKSCDKTHTSPPCS 118 (119) hIgG1cacacatgtccaccgtgctca ccc(p)- gagcccaaatcttgtgacaaaact EPKSCDKTHTSPPCP120 (121) hIgG1 cacacatgtccaccgtgccca sss(p)- gagcccaaatcttctgacaaaactEPKSSDKTHTSPPSP 122 (123) hIgG1 cacacatctccaccgagccca csc(p)-gagcccaaatcttgtgacaaaact EPKSCDKTHTSPPCP 124 (125) hIgG1cacacatctccaccgtgccca ssc(p)- gagcccaaatcttctgacaaaact EPKSSDKTHTSPPCP126 (127) hIgG1 cacacatctccaccgtgccca scc(p)- gagcccaaatcttctgacaaaactEPKSSDKTHTCPPCP 128 (129) hIgG1 cacacatgtccaccgtgccca css(p)-gagcccaaatcttgtgacaaaact EPKSCDKTHTSPPSP 130 (131) hIgG1cacacatctccaccgagccca scs(p)- gagcccaaatcttgtgacaaaact EPKSSDKTHTCPPSP132 (133) hIgG1 cacacatgtccaccgagccca scppcp agttgtccaccgtgccca SCPPCP134 (135) Sequence Identifier (amino acid EFD Nucleotide SequenceAmino acid Sequence sequence) hIgG1 gcacctgaactcctgggtggatcgAPELLGGSSVFLFPPKPKDTLMIS 142 (143) (P238S) tcagtcttcctcttccccccaaaaRTPEVTCVVVDVSHEDPEVKFNWY C_(H2)C_(H3) cccaaggacaccctcatgatctccVDGVEVHNAKTKPREEQYNSTYRV cggacccctgaggtcacatgcgtgVSVLTVLHQDWLNGKEYKCKVSNK gtggtggacgtgagccacgaagacALPAPIEKTISKAKGQPREPQVYT cctgaggtcaagttcaactggtacLPPSRDELTKNQVSLTCLVKGFYP gtggacggcgtggaggtgcataatSDIAVEWESNGQPENNYKTTPPVL gccaagacaaagccgcgggaggagDSDGSFFLYSKLTVDKSRWQQGNV cagtacaacagcacgtaccgtgtgFSCSVMHEALHNHYTQKSLSLSPG gtcagcgtcctcaccgtcctgcac Kcaggactggctgaatggcaaggag tacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaa acaatctccaaagccaaagggcagccccgagaaccacaggtgtacacc ctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacc tgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggag agcaatgggcagccggagaacaactacaagaccacgcctcccgtgctg gactccgacggctccttcttcctctacagcaagctcaccgtggacaag agcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgag gctctgcacaaccactacacgcag aagagcctctccctgtctccgggtaaatga hIgG1 gcacctgaactcctgggtggaccgAPELLGGPSVFLFPPKPKDTLMIS 144 (145) (P331S) tcagtcttcctcttccccccaaaaRTPEVTCVVVDVSHEDPEVKFNWY C_(H2)C_(H3) cccaaggacaccctcatgatctccVDGVEVHNAKTKPREEQYNSTYRV cggacccctgaggtcacatgcgtgVSVLTVLHQDWLNGKEYKCKVSNK gtggtggacgtgagccacgaagacALPASIEKTISKAKGQPREPQVYT cctgaggtcaagttcaactggtacLPPSRDELTKNQVSLTCLVKGFYP gtggacggcgtggaggtgcataatSDIAVEWESNGQPENNYKTTPPVL gccaagacaaagccgcgggaggagDSDGSFFLYSKLTVDKSRWQQGNV cagtacaacagcacgtaccgtgtgFSCSVMHEALHNHYTQKSLSLSPG gtcagcgtcctcaccgtcctgcac Kcaggactggctgaatggcaaggag tacaagtgcaaggtctccaacaaagccctcccagcctccatcgagaaa acaatctccaaagccaaagggcagccccgagaaccacaggtgtacacc ctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacc tgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggag agcaatgggcagccggagaacaactacaagaccacgcctcccgtgctg gactccgacggctccttcttcctctacagcaagctcaccgtggacaag agcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgag gctctgcacaaccactacacgcag aagagcctctccctgtctccgggtaaatga hIgG1 gcacctgaactcctgggtggatcgAPELLGGSSVFLFPPKPKDTLMIS 146 (147) (P238S/ tcagtcttcctcttccccccaaaaRTPEVTCVVVDVSHEDPEVKFNWY P331S) cccaaggacaccctcatgatctccVDGVEVHNAKTKPREEQYNSTYRV C_(H2)C_(H3) cggacccctgaggtcacatgcgtgVSVLTVLHQDWLNGKEYKCKVSNK gtggtggacgtgagccacgaagacALPASIEKTISKAKGQPREPQVYT cctgaggtcaagttcaactggtacLPPSRDELTKNQVSLTCLVKGFYP gtggacggcgtggaggtgcataatSDIAVEWESNGQPENNYKTTPPVL gccaagacaaagccgcgggaggagDSDGSFFLYSKLTVDKSRWQQGNV cagtacaacagcacgtaccgtgtgFSCSVMHEALHNHYTQKSLSLSPG gtcagcgtcctcaccgtcctgcac Kcaggactggctgaatggcaaggag tacaagtgcaaggtctccaacaaagccctcccagcctccatcgagaaa acaatctccaaagccaaagggcagccccgagaaccacaggtgtacacc ctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacc tgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggag agcaatgggcagccggagaacaactacaagaccacgcctcccgtgctg gactccgacggctccttcttcctctacagcaagctcaccgtggacaag agcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgag gctctgcacaaccactacacgcagaagagcctctccctgtctccgggt aaatga Sequence Linker Nucleotide SequenceAmino Acid Sequence Identifier STD1 aattatggtggcggtggctcgggcNYGGGGSGGGGSGGGGSGNS 148 (149) ggtggtggatctggaggaggtggg agtgggaattctSTD2 aattatggtggcggtggctcgggc NYGGGGSGGGGSGGGGSGNYGGGG 150 (151)ggtggtggatctggaggaggtggg SGGGGSGGGGSGNS agtgggaattatggtggcggtggctcgggcggtggtggatctggagga ggtgggagtgggaattct H1 aattct NS 152 (153) H2ggtggcggtggctcggggaattct GGGGSGNS 154 (155) H3 aattatggtggcggtggctctgggNYGGGGSGNS 156 (157) aattct H4 ggtggcggtggctcgggcggtggt GGGGSGGGGSGNS158 (159) ggatctgggaattct H5 aattatggtggcggtggctcgggc NYGGGGSGGGGSGNS160 (161) ggtggtggatctgggaattct H6 ggtggcggtggctcgggcggtggtGGGGSGGGGSGGGGSGNS 162 (163) ggatctgggggaggaggcagcggg aattct H7gggtgtccaccttgtccgaattct GCPPCPNS 164 (165) (G4S) 3ggtggcggtggatccggcggaggt GGGSGGGSGGGS 166 (167) gggtcgggtggcggcggatct(G4S) 4 ggtggcggtggctcgggcggtggt GGGSGGGSGGGSGGGGS 168 (169)ggatctggaggaggtgggagcggg ggaggtggcagt

Example 8 Binding and Functional Studies with Alternative MultispecificFusion Proteins

Experiments that parallel the experiments described above for theprototypical CD20-IgG-CD28 multispecific binding (fusion) molecule wereconducted for each of the additional multivalent binding moleculesdescribed above. In general, the data obtained for these additionalmolecules parallel the results observed for the prototype molecule. Someof the salient results of these experiments are disclosed below. FIG. 14shows results of blocking studies performed on one of the new moleculeswhere both BD1 and BD2 bind to target antigens on the same cell or celltype, in this case, CD20 and CD37. This multispecific, multivalentbinding (fusion) protein was designed with binding domain 1 binding CD20(2H7; VLVH orientation), and binding domain 2 binding CD37, G28-1 VL-VH(LH) or VH-VL (HL). The experiment was performed in order to demonstratethe multispecific properties of the protein.

Blocking Studies: Ramos or BJAB B lymphoblastoid cells (2.5×10⁵) werepre-incubated in 96-well V-bottom plates in staining medium (PBS with 2%mouse sera) with murine anti-CD20 (25 μg/ml) antibody, or murineanti-CD37 (10 μg/ml) antibody, both together or staining medium alonefor 45 minutes on ice in the dark. Blocking antibodies werepre-incubated with cells for 10 minutes at room temperature prior toaddition of the multispecific binding (fusion) protein at theconcentration ranges indicated, usually from 0.02 μg/ml to 10 μg/ml, andincubated for a further 45 minutes on ice in the dark. Cells were washed2 times in staining medium, and incubated for one hour on ice withCaltag (Burlingame, Calif.) FITC goat anti-human IgG (1:100) in stainingmedium, to detect binding of the multispecific binding (fusion) proteinsto the cells. The cells were then washed 2 times with PBS and fixed with1% paraformaldehyde (cat. no. 19943, USB, Cleveland, Ohio). The cellswere analyzed by flow cytometry using a FACsCalibur instrument andCellQuest software (BD Biosciences, San Jose, Calif.). Each data seriesplots the binding of the 2H7-sss-hIgG-STD1-G28-1 HL fusion protein inthe presence of CD20, CD37, or both CD20 and CD37 blocking antibodies.Even though this experiment used one of the cleaved linkers, only thepresence of both blocking antibodies completely eliminates binding bythe multispecific binding (fusion) protein, demonstrating that the bulkof the molecules possess binding function for both CD20 and CD37. Thedata were similar for two cell lines tested in panels A and B, Ramos andBJAB, where the CD20 blocking antibody was more effective than the CD37blocking antibody at reducing the level of binding observed by themultispecific binding (fusion) protein.

ADCC Assays

FIG. 15 shows the results of ADCC assays performed on the CD20-CD37multispecific binding (fusion) proteins. ADCC assays were performedusing BJAB lymphoblastoid B cells as targets and human PBMC as effectorcells. BJAB cells were labeled with 500 μCi/ml ⁵¹Cr sodium chromate (250μCi/μg) for 2 hours at 37° C. in IMDM/10% FBS. The labeled cells werewashed three times in RPMI.10% FBS and resuspended at 4×10⁵ cells/ml inRPMI. Heparinized, human whole blood was obtained from anonymous,in-house donors and PBMC isolated by fractionation over LymphocyteSeparation Media (LSM, ICN Biomedical) gradients. Buffy coats wereharvested and washed twice in RPMI/10% FBS prior to resuspension inRPMI/10% FBS at a final concentration of 5×10⁶ cells/ml. Cells werecounted by trypan blue exclusion using a hemacytometer prior to use insubsequent assays. Reagent samples were added to RPMI medium with 10%FBS at 4 times the final concentration and three 10 fold serialdilutions for each reagent were prepared. These reagents were then addedto 96-well U-bottom plates at 50 μl/well for the indicated finalconcentrations. The ⁵¹Cr-labeled BJAB cells were added to the plates at50 μl/well (2×10⁴ cells/well). The PBMCs were then added to the platesat 100 μl/well (5×10⁵ cells/well) for a final ratio of 25:1 effector(PBMC):target (BJAB). Effectors and targets were added to medium aloneto measure background killing. The ⁵¹Cr-labeled cells were added tomedium alone to measure spontaneous release of ⁵¹Cr and to medium with5% NP40 (cat. no. 28324, Pierce, Rockford, Ill.) to measure maximalrelease of ⁵¹Cr. Reactions were set up in triplicate wells of a 96-wellplate. Multispecific binding (fusion) proteins were added to wells at afinal concentration ranging from 0.01 μg/ml to 10 μg/ml, as indicated onthe graphs. Each data series plots a different multispecific binding(fusion) protein or the corresponding single specificity SMIPs at thetitration ranges described. Reactions were allowed to proceed for 6hours at 37° C. in 5% CO₂ prior to harvesting and counting. Twenty-fiveμl of the supernatant from each well were then transferred to a LumaPlate 96 (cat. no. 6006633, Perkin Elmer, Boston, Mass.) and driedovernight at room temperature. CPM released was measured on a PackardTopCounNXT. Percent specific killing was calculated by subtracting (cpm{mean of triplicate samples} of sample−cpm spontaneous release)/(cpmmaximal release-cpm spontaneous release)×100. Data are plotted as %specific killing versus protein concentration. The data demonstrate thatthe multispecific binding (fusion) protein is able to mediate ADCCactivity against cells expressing the target antigen(s) as well as thesingle specificity SMIPs for CD20 and/or CD37, but does not showaugmentation in the level of this effector function.

Co-Culture Experiments

FIG. 16 shows the results of experiments designed to look at otherproperties of this type of multispecific binding (fusion) protein, wherehaving two binding domains against targets expressed on the same cell orcell type might result in synergistic effects by signaling/bindingthrough the two surface receptors bound. The co-culture experiments wereperformed using PBMC isolated as described for the ADCC assays above.These PBMC were resuspended in culture medium at 2×10⁶ cells/ml in afinal volume of 500 μl/well, and cultured alone or incubated with singlespecificity SMIPs for CD20, CD37, CD20+CD37, or the multispecificbinding (fusion) protein using the H7 linker, [2H7-sss-IgG-H7-G28-1 HL].Each of the test reagents was added at a final concentration of 20μg/ml. After 24 hours of culture, no real differences were seen in the %of B cells in culture; however, when the cells were subjected to flowcytometry, cell clumping was visible in the FWD X 90 staining patternfor the cultures containing the multispecific binding (fusion) protein,indicating that the B cells expressing the two target antigens wereengaged in homotypic adhesion. After 72 hours in culture, themultispecific binding (fusion) protein resulted in the death of almostall the B cells present. The combination of the two single-specificitySMIPs also drastically decreased the percentage of B cells, but not tothe level seen with the multispecific binding molecule. These datasuggest that engaging both binding domains for CD20 and CD37 on the samemultispecific molecule, results in homotypic adhesion between B cellsand may also result in binding of both CD20 and CD37 antigens on thesame cell. Without wishing to be bound by theory, the synergistic effectin eliminating target cells may be due (1) to the binding throughbinding domains 1 and 2 on the same cell types, and/or (2) tointeractions of the effector function domain (constant sub-region) ofthe multivalent binding molecules with monocytes or other cell types inthe PBMC culture that result in delayed killing. The kinetics of thiskilling effect are not rapid, taking more than 24 hours to be achieved,indicating that it is may be a secondary effect, requiring production ofcytokines or other molecules prior to the effects being observed.

Apoptosis Assays

FIG. 17 shows the results of experiments designed to explore theinduction of apoptosis after treatment of B cell lines with either the[2H7-sss-hIgG-H7-G28-1 HL] multispecific, multivalent binding (fusion)proteins or the single specificity CD20 and/or CD37 SMIPS, alone and incombination with one another. Ramos cells (panel A; ATCC No. CRL-1596),and Daudi cells (panel B; ATCC No. CCL-213) were incubated overnight (24hours) at 37° C. in 5% CO₂ in Iscoves (Gibco) complete medium with 10%FBS at 3×10⁵ cells/ml and 5, 10, or 20 μg/ml fusion proteins. Forcombination experiments with the single specificity SMIPs, the proteinswere used at the following concentrations: TRU-015 (CD20 directedSMIP)=10 μg/ml, with 5 μg/ml G28-1 LH (CD37 directed SMIP).Alternatively, TRU-015=20 μg/ml was combined with G28-1 LH at 10 μg/ml.Cells were then stained with Annexin V-FITC and propidium iodide usingthe BD Pharmingen Apoptosis Detection Kit I cat. no. 556547), andprocessed according to kit instructions. The cells were gently vortexed,incubated in the dark at room temperature for 15 minutes, and diluted in400 μl binding buffer prior to analysis. Samples were analyzed by flowcytometry on a FACsCalibur (Becton Dickinson) instrument using CellQuest software (Becton Dickinson). The data are presented as columnargraphs plotting the percentage of Annexin V/propidium iodide positivecells versus type of treatment. Clearly, the multispecific binding(fusion) protein is able to induce a significantly higher level ofapoptotic death in both cell lines than the single specificity reagents,even when used together. This increased functional activity reflects aninteraction of the coordinate binding of BD1 and BD2 (specific for CD20and CD37) receptors on the target cells.

Example 9 Binding and Functional Properties of 2H7-hIgG-G19-4Multispecific Binding (Fusion) Proteins

This example describes the binding and functional properties of the2H7-hIgG-G19-4 multispecific fusion proteins. The construction of thesemolecules is described in Example 7. Expression and purification are asdescribed in previous Examples.

Binding experiments were performed as described for previous molecules,except that the target cells used to measure CD3 binding were Jurkatcells expressing CD3 on their surface. Refer to FIG. 18, where the topgraph shows binding curves obtained for binding of the CD20-CD3multispecific molecules to Jurkat cells using purified proteins seriallydiluted from 20 to 0.05 μg/ml. The HL orientation of the G19-4specificity seems to bind better to the CD3 antigen than does the LHorientation. The lower panel shows the binding curves obtained for theBD1, the binding domain recognizing CD20. All of the molecules bindwell, and at a level nearly equivalent to a single specificity SMIP forCD20.

ADCC Assays

For the data presented in FIG. 19, ADCC assays were performed asdescribed in the previous Example. In this case, the fusion proteinswere all 2H7-hIgG-G19-4 variants or combinations of thesingle-specificity SMIPs (2H7, specific for CD20) or antibodies (G19-4,specific for CD3). In addition, for the data presented in the lowerpanel of FIG. 19, NK cells were depleted from PBMC prior to use, bymagnetic bead depletion using a MACS (Miltenyi Biotec, Auburn, Calif.)column separation apparatus and NK cell-specific CD16 magneticmicrobeads (cat no.: 130-045-701). The data presented in the two panelsdemonstrate that all of the CD20-hIgG-CD3 multispecific moleculesmediate ADCC, regardless of whether NK cells are depleted or total PBMCare used in the assay. For the TRU 015 or combinations of G19-4 andTRU015, only cultures containing NK cells could mediate ADCC. G19-4 didnot work well in either assay against BJAB targets, which do not expressCD3, although G19-4 may have bound to CD3 expressing NK T cells andactivated these cells in the first assay shown. The killing observed inthe lower panel for the multispecific binding (fusion) proteins isprobably mediated through activation of cytotoxicity in the T cellpopulation by binding CD3, against the BJAB targets expressing the CD20antigen. This killing activity appears to be relatively insensitive tothe dosage of the molecules over the concentration ranges used, and isstill significantly different from the other molecules tested, even at aconcentration of 0.01 ug/ml.

Example 10 Multivalent Binding Molecules

Other embodiments include linker domains derived from immunoglobulins.More specifically, the source sequences for these linkers are sequencesobtained by comparing regions present between the V-like domains or theV- and C-like domains of other members of the immunoglobulinsuperfamily. Because these sequences are usually expressed as part ofthe extracellular domain of cell surface receptors, they are expected tobe more stable to proteolytic cleavage, and should also not beimmunogenic. One type of sequence that is not expected to be as usefulin the role of a linker for the multivalent binding (fusion) proteins isthe type of sequence expressed on surface-expressed members of the -Igsuperfamily, but that occur in the intervening region between the C-likedomain and the transmembrane domain. Many of these molecules have beenobserved in soluble form, and are cleaved in these intervening regionsclose to the cell membrane, indicating that the sequences are moresusceptible to cleavage than the rest of the molecule.

The linkers described above are inserted into either a singlespecificity SMIP, between the binding domain and the effector functiondomain, or are inserted into one of the two possible linker positions ina multivalent binding (fusion) protein, as described herein.

A complete listing of the sequences disclosed in this application isappended, and is incorporated herein by reference in its entirety. Thecolor coding indicating the sequence of various regions or domains ofthe particular polynucleotides and polypeptides are useful inidentifying a corresponding region or domain in the sequence of any ofthe molecules disclosed herein.

Example 11 Screening Matrix for Scorpion Candidates Targeting B-Cells

Introduction

As a means of identifying combinations of paired monoclonal antibodybinding domains that would most likely yield useful and potentmultivalent binding molecules, or scorpions, against a targetpopulation, a series of monoclonal antibodies against B cell antigenswas tested in a combination matrix against B cell lines representingvarious non Hodgkin's lymphomas. To ensure that all possible pairwisecomparisons of antibodies known or expected to bind to the cell ofinterest are assayed, a two-dimensional matrix of antibodies may be usedto guide the design of studies using a given cell type. Monoclonalantibodies against numerous B cell antigens known by their clusterdesignations (CDs) are recorded in the left column. Some of theseantibodies (designated by the antigen(s) to which they specificallybind), i.e., CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72,CD79a, CD79b, CD80, CD81, CD86, and CL II (MHC Class II), wereincubated, alone or in combination with other members of this monoclonalantibody set, with antigen-positive target cells. The variable domainsof these antibodies are contemplated as binding domains in exemplaryembodiments of the multivalent binding molecules. Using the knowledge inthe art and routine procedures, those of skill in the art are able toidentify suitable antibody sequences (nucleic acid encoding sequences aswell as amino acid sequences), for example in publicly availabledatabases, to generate a suitable antibody or fragment thereof (e.g., byhybridization-based cloning, PCR, peptide synthesis, and the like), andto construct multivalent binding molecules using such compounds. Sourcesof exemplary antibodies from which binding domains were obtained asdescribed herein are provided in Table 6. Typically, a cloning orsynthesis strategy that realizes the CDR regions of an antibody chainwill be used, although any antibody, fragment thereof, or derivativethereof that retains the capacity to specifically bind to a targetantigen is contemplated.

Stated in more detail, the cloning of heavy and/or light chain variableregions of antibodies from hybridomas is standard in the art. There isno requirement that the sequence of the variable region of interest beknown in order to obtain that region using conventional cloningtechniques. See, e.g., Gilliland et al., Tissue Antigens 47(1):1-20(1996). To prepare single-chain polypeptides comprising a variableregion recognizing a murine or human leukocyte antigen, a method wasdevised for rapid cloning and expression that yielded functional proteinwithin two to three weeks of RNA isolation from hybridoma cells.Variable regions were cloned by poly-G tailing the first-strand cDNAfollowed by anchor PCT with a forward poly-C anchor primer and a reverseprimer specific for the constant region sequence. Both primers containflanking restriction endonuclease sites for insertion into pUC19. Setsof PCR primers for isolation of murine, hamster and rat V_(L) and V_(H)genes were generated. Following determination of consensus sequences fora specific V_(L) and V_(H) pair, the V_(L) and V_(H) genes were linkedby DNA encoding an intervening peptide linker (typically encoding(Gly₄Ser)₃) and the V_(L)-linker-V_(H) gene cassettes were transferredinto the pCDM8 mammalian expression vector. The constructs weretransfected into COS cells and sFvs were recovered from conditionedculture medium supernatant. This method has been successfully used togenerate functional sFv to human CD2, CD3, CD4, CD8, CD28, CD40, CD45and to murine CD3 and gp39, from hybridomas producing murine, rat, orhamster antibodies. Initially, the sFvs were expressed as fusionproteins with the hinge-C_(H2)-C_(H3) domains of human IgG1 tofacilitate rapid characterization and purification using goat anti-humanIgG reagents or protein A. Active sFv could also be expressed with asmall peptide, e.g., a tag, or in a tailless form. Expression of CD3 (G19-4) sFv tailless forms demonstrated increased cellular signalingactivity and revealed that sFvs have potential for activating receptors.

Alternatively, identification of the primary amino acid sequence of thevariable domains of monoclonal antibodies can be achieved directly,e.g., by limited proteolysis of the antibody followed by N-terminalpeptide sequencing using, e.g., the Edman degradation method or byfragmentation mass spectroscopy. N-terminal sequencing methods are wellknown in the art. Following determination of the primary amino acidsequence, the variable domains, a cDNA encoding this sequence isassembled by synthetic nucleic acid synthesis methods (e.g., PCR)followed by scFv generation. The necessary or preferred nucleic acidmanipulation methods are standard in the art.

Fragments, derivatives and analogs of antibodies, as described above,are also contemplated as suitable binding domains. Further, any of theconstant sub-regions described above are contemplated, includingconstant sub-regions comprising any of the above-described hingeregions. Additionally, the multivalent single-chain binding moleculesdescribed in this example may include any or all of the linkersdescribed herein.

Monoclonal antibodies were initially exposed to cells and thencross-linked using a goat anti-mouse second-step antibody (2^(nd) step).Optionally, one could cross-link the antibodies prior to contactingcells with the antibodies, e.g., by cross-linking the antibodies insolution. As another alternative, monoclonal antibodies could becross-linked in a solid phase by adsorbing onto the plastic bottom oftissue culture wells or “trapped” on this plastic by means of goatanti-mouse antibody adsorbed to the plastic, followed by plate-basedassays to evaluate, e.g., growth arrest or cell viability.

Inversion of phosphatidylserine from the cytosolic side of the cellmembrane to the exterior cell surface of that plasma membrane is anaccepted indicator of pro-apoptotic events. Progression to apoptosisleads to loss of cell membrane integrity, which can be detected by entryof a cell-impermeant intercalating dye, e.g., propidium iodide (PI).Following cell exposure to monoclonal antibodies alone or incombination, a dual, pro-apoptotic assay was performed and treated cellpopulations were scored for cell surface-positive annexin V (ANN) and/orPI inclusion.

Annexin V Binding/Propidium Iodide Internalization Analysis

Cells and cell culture conditions. Experiments were performed to examinethe effect of cross-linking two different monoclonal antibodies againsttargets expressed on four human B-cell lines. Effects on cell lines weremeasured by determining levels of ANN and/or PI staining followingexposure. The human B cell lines BJAB, Ramos (ATCC#CRL-1596), Daudi(ATCC#CCL-213), and DHL-4 (DSMZ#ACC495) were incubated for 24 hours at37° C. in 5% CO₂ in Iscoves (Gibco) complete medium with 10% FBS. Cellswere maintained at a density between 2-8×10⁵ cells/ml and a viabilitytypically >95% prior to study.

Experiments were conducted at a cell density of 2×10⁵ cells/ml and 2μg/ml of each comparative monoclonal antibody from a matrix againstB-cell antigens. Each comparator monoclonal antibody was added at 2μg/ml alone or individually when combined with each matrix monoclonalantibody, also at 2 μg/ml. Table 6 lists the catalog number and sourcesof monoclonal antibodies used in these experiments. For cross-linkingthese monoclonal antibodies in solution, goat anti-mouse IgG (JacksonLabs catalog no. 115-001-008) was added to each well at a concentrationratio of 2:1 (goat anti-mouse: each monoclonal antibody), e.g., a wellwith only one monoclonal antibody at 2 μg/ml would have goat anti-mouseadded to a final concentration of 4 μg/ml, while wells with bothcomparator monoclonal antibody (2 μg/ml) and a monoclonal antibody fromthe matrix (2 μg/ml) would have 8 μg/ml of goat anti-mouse antibodyadded to the well.

After 24 hours of incubation at 37° C. in 5% CO₂, cells were stainedwith Annexin V-FITC and propidium iodide using the BD Pharmingen AnnexinV-FITC Apoptosis Detection Kit I (#556547). Briefly, cells were washedtwice with cold PBS and resuspended in “binding buffer” at 1×10⁶cells/ml. One hundred microliters of the cells in binding buffer werethen stained with 5 μl of Annexin V-FITC and 5 μl of propidium iodide.The cells were gently mixed and incubated in the dark at roomtemperature for 15 minutes. Four hundred microliters of binding bufferwere then added to each sample. The samples were then read on aFACsCalibur (Becton Dickinson) and analyzed using Cell Quest software(Becton Dickinson).

TABLE 6 Table 6. Antibodies against B cell antigens used in this studyand their sources. Name Catalog number Commercial supplier Anti-CD19#C2269-74 US Biological (Swampscott, MA) Anti-CD20 #169-820 Ancell Corp(Bayport, MN) Anti-CD21 #170-820 Ancell Corp (Bayport, MN) Anti-CD22#171-820 Ancell Corp (Bayport, MN) Anti-CD23 #172-820 Ancell Corp(Bayport, MN) Anti-CD30 #179-820 Ancell Corp (Bayport, MN) Anti-CD37#186-820 Ancell Corp (Bayport, MN) Anti-CD40 #300-820 Ancell Corp(Bayport, MN) Anti-CD70 #222-820 Ancell Corp (Bayport, MN) Anti-CD72#C2428-41B1 US Biological (Swampscott, MA) Anti-CD79a #235-820 AncellCorp (Bayport, MN) Anti-CD79b #301-820 Ancell Corp (Bayport, MN)Anti-CD80 #110-820 Ancell Corp (Bayport, MN) Anti-CD81 #302-820 AncellCorp (Bayport, MN) Anti-CD86 #307-820 Ancell Corp (Bayport, MN) Anti-CLII DR, #131-820 Ancell Corp (Bayport, MN) DQ, DP

Addition of the cross-linking antibody (e.g., goat anti-mouse antibody)to monoclonal antibody A alone resulted in increased cell sensitivity,suggesting that a multivalent binding molecule, or scorpion, constructedwith two binding domains recognizing the same antigen would be effectiveat increasing cell sensitivity. Without wishing to be bound by theory,this increased sensitivity could be due to antigen clustering andaltered signaling. TNF receptor family members, for example, requirehomo-multimerization for signal transduction and scorpions withequivalent binding domains on each end of the molecule could facilitatethis interaction. The clustering and subsequent signaling by CD40 is anexample of this phenomenon in the B cell system.

As shown in FIGS. 20, 21 and 22, the addition of monoclonal antibody Aand monoclonal antibody B against different antigens will produceadditive or in some combinations greater than additive (i.e.,synergistic) pro-apoptotic effects on treated cells. In FIG. 20, forexample, the combination of anti-CD20 with monoclonal antibodies againstother B cell antigens all resulted, to varying extents, in increasedcell sensitivity. Some combinations, such as anti-CD20 combined withanti-CD19 or anti-CD20 combined with anti-CD21, however, producedgreater than additive pro-apoptotic effects, indicating that multivalentbinding molecules or scorpions composed of these binding domains shouldbe particularly effective at eliminating transformed B cells. Referringto FIG. 20, the percentage of cells exhibiting pro-apoptotic activitieswhen exposed to anti-CD 20 antibody alone is about 33% (verticallystriped bar corresponding to “20,” i.e., the anti-CD20 antibody); thepercentage of pro-apoptotic cells upon exposure to anti-CD19 antibody isabout 12% (vertically striped bar in FIG. 20 corresponding to “19,”i.e., the anti-CD19 antibody); and the percentage of pro-apoptotic cellsupon exposure to both anti-CD20 and anti-CD19 antibodies is about 73%(horizontally striped bar in FIG. 20 corresponding to “19”). The 73% ofpro-apoptotic cells following exposure to both antibodies issignificantly greater than the 45% (33%+12%) sum of the effectsattributable to each individual antibody, indicating a synergisticeffect attributable to the anti-CD19 and anti-CD 20 antibody pair.Useful multivalent binding molecules include molecules in which the twobinding domains lead to an additive effect on B-cell behavior as well asmultivalent binding molecules in which the two binding domains lead tosynergistic effects on B-cell behavior. In some embodiments, one bindingdomain will have no detectable effect on the measured parameter of cellbehavior, with each of the paired binding domains contributing todistinct aspects of the activities of the multivalent binding molecule,such as a multispecific, multivalent binding molecule (e.g., bindingdomain A binds to a target cell and promotes apoptosis while bindingdomain B binds to a soluble therapeutic such as a cytotoxin). Dependingon the design of a multivalent binding molecule, the issue of the typeof combined effect (additive, synergistic, or inhibitory) of the twobinding domains on a target cell may not be relevant because one of thebinding domains is specific for a non-cellular (e.g., soluble) bindingpartner or is specific for a cell-associated binding partner, but on adifferent cell type.

Exemplary binding domain pairings producing additive, synergistic orinhibitory effects, as shown in FIGS. 20-23, are apparent from Tables 7and 8. Table 7 provides quantitative data extracted from each of FIGS.20-23 in terms of the percentage of cells staining positive for ANNand/or PI. Table 8 provides calculations using the data of Table 7 thatprovided a basis for determining whether the interaction of a given pairof antibodies yielded an additive, synergistic, or inhibitory effect,again as assessed by the percentage of cells staining positive for ANNand/or PI.

TABLE 7 Name Anti-CD20 Anti-CD79b Anti-CL II Anti-CD22 Anti-CD19 13/73*18/76/66 14/47/46 12/11 Anti-CD20 33/NA 42/94/92 33/71/76 28/33Anti-CD21 14/75 22/50/76 18/24/40 11/11 Anti-CD22  8/55 12/39/3312/19/17 10/12 Anti-CD23  8/41 12/63/55 14/22/17 10/12 Anti-CD30  8/3814/72/61 12/56/61 10/11 Anti-CD37 15/45 19/92/86 20/60/62 19/20Anti-CD40 10/48 12/44/30 13/21/28 14/13 Anti-CD70  9/40 12/56/3915/21/15 10/10 Anti-CD72 NA 16/60/64 30/78/63 17/17 Anti-CD79a 21/6643/42/50 28/55/51 14/14 Anti-CD79b 46/88 70/70/68 45/80/76 26/16Anti-CD80  7/41 14/35/30 15/19/17 11/11 Anti-CD81 14/65 25/86/8325/54/43 19/20 Anti-CD86  7/38 16/58/42 15/24/18 14/11 Anti-CL II 53/7752/96/98 47/52/43 72/57 *In columns 2-4 of Table 7, the numerical valuesreflect the heights of histogram bars in FIGS. 20-22, respectively, withthe first number in each cell denoting the height of a verticallystriped bar, the second number denoting the height of a horizontallystriped bar and, where present, the third number reflecting the heightof a stippled bar. In column 5, the first number reflects the height ofa solid bars and the second number reflects the height of aslant-striped bar in FIG. 23.

TABLE 8 Name Anti-CD20 Anti-CD79b Anti-CL II Anti-CD22 Anti-CD19 S: 13 +33 = 46* A: 18 + 56 = 74 S: 14 + 26 = 40 I: 12 + 10 = 22 A: 18 + 43 = 61S: 14 + 18 = 32 Anti-CD20 NA A: 42 + 56 = 98 S: 33 + 26 = 59 A/I: 28 +10 = 38 A: 42 + 43 = 85 S: 33 + 18 = 51 Anti-CD21 S: 14 + 33 = 47 I:22 + 56 = 78 I: 18 + 26 = 44 I: 11 + 10 = 21 S: 22 + 43 = 65 A: 18 + 18= 36 Anti-CD22 S: 8 + 33 = 41 I: 12 + 56 = 68 I: 12 + 26 = 38 NA I: 12 +43 = 55 I: 12 + 18 = 30 Anti-CD23 A: 8 + 33 = 41 A: 12 + 56 = 68 I: 14 +26 = 40 I: 10 + 10 = 20 A: 12 + 43 = 55 I: 14 + 18 = 32 Anti-CD30 A: 8 +33 = 41 A: 14 + 56 = 70 S: 12 + 26 = 38 I: 10 + 10 = 20 A: 14 + 43 = 57S: 12 + 18 = 30 Anti-CD37 A: 15 + 33 = 48 S: 19 + 56 = 75 S: 20 + 26 =46 I: 19 + 10 = 29 S: 19 + 43 = 62 S: 20 + 18 = 38 Anti-CD40 A/S: 10 +33 = 43 I: 12 + 56 = 68 I: 13 + 26 = 39 I: 14 + 10 = 24 I: 12 + 43 = 55A: 13 + 18 = 31 Anti-CD70 A: 9 + 33 = 42 I: 12 + 56 = 68 I: 15 + 26 = 41I: 10 + 10 = 20 I: 12 + 43 = 55 I: 15 + 18 = 33 Anti-CD72 NA I: 16 + 56= 72 S: 30 + 26 = 56 I: 17 + 10 = 27 A: 16 + 43 = 59 S: 30 + 18 = 48Anti-CD79a S: 21 + 33 = 54 I: 43 + 56 = 99 A: 28 + 26 = 54 I: 14 + 10 =24 I: 43 + 43 = 86 A: 28 + 18 = 46 Anti-CD79b S: 46 + 33 = 79 NA S: 45 +26 = 71 I: 26 + 10 = 36 S: 45 + 18 = 63 Anti-CD80 A: 7 + 33 = 40 I: 14 +56 = 70 I: 15 + 26 = 41 I: 11 + 10 = 21 I: 14 + 43 = 57 I: 15 + 18 = 33Anti-CD81 S: 14 + 33 = 47 A: 25 + 56 = 81 A: 25 + 26 = 51 I: 19 + 10 =29 S: 25 + 43 = 68 A: 25 + 18 = 43 Anti-CD86 A: 7 + 33 = 40 I: 16 + 56 =72 I: 15 + 26 = 41 I: 14 + 11 = 25 I: 16 + 43 = 59 I: 15 + 18 = 33Anti-CL II I: 53 + 33 = 86 A: 52 + 56 = 108 NA I: 72 + 10 = 82 A: 52 +43 = 95 “A” means an “additive” effect was observed “S” means a“synergistic” effect was observed “I” means an “inhibitory” effect wasobserved *Equation schematic: A + B = C, where “A” is the percent ANNand/or P1 positive cells due to matrix antibody alone, “B” is thepercent ANN and/or PI positive cells due to the common antibody(anti-CD20 for FIG. 20, anti-CD79b for FIG. 21, anti-CLII for FIG. 22,and anti-CD22 for FIG. 23), and “C” is the expected additive effect.(See Table 7, above, for the quantitative data corresponding to FIGS.20-23.) Where two equations are present in a cell, the upper equationreflects results use of the higher indicated concentration of commonantibody; the lower equation reflects use of the lower indicatedconcentration of common antibody.

In some embodiments, the two binding domains interact in an inhibitory,additive or synergistic manner in sensitizing (or de-sensitizing) atarget cell such as a B cell. FIG. 23 shows the protective, orinhibitory, effects resulting from combining anti-CD22 antibody withstrongly pro-apoptotic monoclonal antibodies such as the anti-CD79bantibody or anti-MHC class II (i.e., anti-CL II) antibody. For example,FIG. 23 and Table 7 show that anti-CD22 antibody alone induces no morethan about 10% of cells to exhibit pro-apoptotic behavior (solid barcorresponding to “22” in FIG. 23) and anti-CD79b induces about 26%pro-apoptotic cells (solid bar corresponding to “CD79b” in FIG. 23). Incombination, however, anti-CD22 and anti-CD79b induce only about 16%pro-apoptotic cells (slant-striped bar corresponding to “79b” in FIG.23). Thus, the combined antibodies induce 16% pro-apoptotic cells, whichis less than the 38% sum of the individual effects attributable toanti-CD22 (12%) and anti-CD79b (26%). Using this approach, an inspectionof FIG. 23 and/or Tables 7-8 reveals that anti-CD22 antibody, and byextension a multispecific, multivalent binding molecule comprising ananti-CD22 binding domain, when used in separate combination with each ofthe following antibodies (or corresponding binding domains): anti-CD19,anti-CD20, anti-CD21, anti-CD23, anti-CD30, anti-CD37, anti-CD40,anti-CD70, anti-CD72, anti-CD79a, anti-CD79b, anti-CD80, anti-CD81,anti-CD86 and anti-MHC class II antibodies/binding domains, will resultin an inhibited overall effect.

Without wishing to be bound by theory, the data can be interpreted asindicating that anti-CD22 antibody, or a multispecific, multivalentbinding molecule comprising an anti-CD22 binding domain, will protectagainst, or mitigate an effect of, any of the antibodies listedimmediately above. More generally, a multispecific, multivalent bindingmolecule comprising an anti-CD22 binding domain will inhibit the effectarising from interaction with any of CD19, CD20, CD21, CD23, CD30, CD37,CD40, CD70, CD72, CD 79a, CD79b, CD80, CD81, CD86, and MHC class IImolecules. It can be seen in FIG. 23 and Table 8 that anti-CD22antibody, and by extension a binding domain comprising an anti-CD22binding domain, will function as an inhibitor or mitigator of theactivity of any antibody/binding domain recognizing a B-cell surfacemarker such as a CD antigen. Multivalent binding molecules, includingmultispecific, multivalent binding molecules, are expected to be usefulin refining treatment regimens for a variety of diseases wherein theactivity of a binding domain needs to be attenuated or controlled.

In addition to the inhibitory, additive or synergistic combined effectof two binding domains interacting with a target cell, typically throughthe binding of cell-surface ligands, the experimental results disclosedherein establish that a given pair of binding domains may provide adifferent type of combined effect depending on the relativeconcentrations of the two binding domains, thereby increasing theversatility of the invention. For example, Table 8 discloses thatanti-CD21 and anti-CD79b interact in an inhibitory manner at the highertested concentration of anti-CD79b, but these two antibodies interact ina synergistic manner at the lower tested concentration of anti-CD79b.Although some embodiments will use a single type of multivalent bindingmolecule, i.e., a monospecific, multivalent binding molecule,comprising, e.g., a single CD21 binding domain and a single CD79bbinding domain, the invention comprehends mixtures of multivalentbinding molecules that will allow adjustments of relative binding domainconcentrations to achieve a desired effect, such as an inhibitory,additive or synergistic effect. Moreover, the methods of the inventionencompass use of a single multivalent binding molecule in combinationwith another binding molecule, such as a conventional antibody molecule,to adjust or optimize the relative concentrations of binding domains.Those of skill in the art will be able to determine useful relativeconcentrations of binding domains using standard techniques (e.g., bydesigning experimental matrices of two dilution series, one for eachbinding domain).

Without wishing to be bound by theory, it is recognized that the bindingof one ligand may induce or modulate the surface appearance of a secondligand on the same cell type, or it may alter the surface context of thesecond ligand so as to alter its sensitivity to binding by a specificbinding molecule such as an antibody or a multivalent binding molecule.

Although exemplified herein using B cell lines and antigens, thesemethods to determine optimally effective multivalent binding molecules(i.e., scorpions) are applicable to other disease settings and targetcell populations, including other normal cells, their aberrant cellcounterparts including chronically stimulated hematopoietic cells,carcinoma cells and infected cells.

Other signaling phenotypes such as Ca²⁺ mobilization; tyrosinephosphoregulation; caspase activation; NF-κB activation; cytokine,growth factor or chemokine elaboration; or gene expression (e.g., inreporter systems) are also amenable to use in methods of screening forthe direct effects of monoclonal antibody combinations.

As an alternative to using a secondary antibody to cross-link theprimary antibodies and mimic the multivalent binding molecule orscorpion structure, other molecules that bind the Fc portion ofantibodies, including soluble Fc receptors, protein A, complementcomponents including C1q, mannose binding lectin, beads or matricescontaining reactive or cross-linking agents, bifunctional chemicalcross-linking agents, and adsorption to plastic, could be used tocross-link multiple monoclonal antibodies against the same or differentantigens.

Example 12 Multivalent Binding Protein with Effector Function, orScorpion, Structures

The general schematic structure of a scorpion polypeptide is H2N-bindingdomain 1-scorpion linker-constant sub-region-binding domain 2. scorpionsmay also have a hinge-like region, typically a peptide region derivedfrom an antibody hinge, disposed N-terminal to binding domain 1. In somescorpion embodiments, binding domain 1 and binding domain 2 are eachderived from an immunoglobulin binding domain, e.g., derived from aV_(L) and a V_(H). The V_(L) and a V_(H) are typically joined by alinker. Experiments have been conducted to demonstrate that scorpionpolypeptides may have binding domains that differ from an immunoglobulinbinding domain, including an Ig binding domain from which the scorpionbinding domain was derived, by amino acid sequence differences thatresult in a sequence divergence of typically less than 5%, andpreferably less than 1%, relative to the source Ig binding domain.

Frequently, the sequence differences result in single amino acidchanges, such as substitutions. A preferred location for such amino acidchanges is in one or more regions of a scorpion binding domain thatcorrespond, or exhibit at least 80% and preferably 85% or 90%, sequenceidentity to an Ig complementarity determining region (CDR) of an Igbinding domain from which the scorpion binding domain was derived.Further guidance is provided by comparing models of peptides binding thesame target, such as CD20. With respect to CD20, epitope mapping hasrevealed that the 2H7 antibody, which binds CD20, recognizes theAla-Asn-Pro-Ser (ANPS) motif of CD20 and it is expected thatCD20-binding scorpions will also recognize this motif. Amino acidsequence changes that result in the ANPS motif being deeply embedded ina pocket formed of scorpion binding domain regions corresponding to IgCDRs are expected to be functional binders of CD20. Modeling studieshave also revealed that scorpion regions corresponding to CDR3 (V_(L)),CDR1-3 (V_(H)) contact CD20 and changes that maintain or facilitatethese contacts are expected to yield scorpions that bind CD20.

In addition to facilitating interaction of a scorpion with its target,changes to the sequences of scorpion binding domains (relative tocognate Ig binding domain sequences) that promote interaction betweenscorpion binding domain regions that correspond to Ig V_(L) and V_(H)domains are contemplated. For example, in a CD20-binding scorpion regioncorresponding to V_(L), the sequence SYN may be changed by substitutingan amino acid for Val (V33), such as His, resulting in the sequenceSYIH. This change is expected to improve interaction between scorpionregions corresponding to V_(L) and V_(H) domains. Further, it isexpected that the addition of a residue at the N-terminus of a scorpionregion corresponding to V_(H)-CDR3 will alter the orientation of thatscorpion region, likely affecting its binding characteristics, becausethe N-terminal Ser of V_(H)-CDR3 makes contact with CD20. Routine assayswill reveal those orientations that produce desirable changes in bindingcharacteristics. It is also contemplated that mutations in scorpionregions corresponding to V_(H)-CDR2 and/or V_(H)-CDR3 will createpotential new contacts with a target, such as CD20. For example, basedon modeling studies, it is expected that substitutions of either Y105and W106 (found in the sequence NSYW) in a region corresponding toV_(H)-CDR3 will alter the binding characteristics of a scorpion in amanner amenable to routine assay for identifying scorpions with modifiedbinding characteristics. By way of additional example, it is expectedthat an alteration in the sequence of a scorpion binding domaincorresponding to an Ig VL-CDR3, such as the Trp (W) in the sequenceCQQW, will affect binding. Typically, alterations in a scorpion regioncorresponding to an Ig CDR will be screened for those scorpionsexhibiting an increase in affinity for the target.

Based on the model structure of the humanized CD20 scFv binding domain20-4, on the published information relating to the CD20 extracellularloop structure (Du, et al., J Biol. Chem. 282(20):15073-80 (2007)), andon the CD20 binding epitope recognized by the mouse 2H7 antibody (whichwas the source of CDRs for the humanized 20-4 scFv binding domain),mutations were engineered in the CDR regions of the 2Lm20-4×2Lm20-4scorpion with the aim of improving the affinity of its binding to CD20.First, the mutations were design to influence the 20-4 CDR conformationand to promote more efficient binding to the CD20 extracellular loop.Second, the introduced changes were designed to provide newintermolecular interactions between the 2Lm20-4×2Lm20-4 scorpion and itstarget. These mutations include: VL CDR1V33H i.e., a substitution of Hisfor Val at position 33 of CDR1 in the VL region), VL CDR3 W90Y, VH CDR2D57E, VH CDR3 insertion of V after residue S99, VH CDR3 Y101K, VH CDR3N103G, VH CDR3 N104G, and VH CDR3Y105D. Due to expected synergisticeffects of combining some of theses mutations, 11 mutants were designed,combining different mutations as shown in Table 9 (residues introducedby mutation are bolded and underscored).

TABLE 9 V_(L )CDR1 V_(L )CDR3 V_(H )CDR2 V_(H )CDR3 RASSSVSYI HQQWSFNPPT AIYPGNGDTSYNQK SV YYSNYWYFDL FKG RASSSVSYI H QQWSFNPPTAIYPGNGDTSYNQK SV YY GG YWYFDL FKG RASSSVSYI H QQWSFNPPT AIYPGNGDTSYNQKSYYSNS D WYFDL FKG RASSSVSYI H QQWSFNPPT AIYPGNGDTSYNQK SYYS GGD WYFDLFKG RASSSVSYIV QQWSFNPPT AIYPGNGDTSYNQK SY K SNSYWYFDL FKG RASSSVSYIVQQWSFNPPT AIYPGNG E TSYNQK SYYSNSYWYFDL FKG RASSSVSYIV QQ Y SFNPPTAIYPGNGDTSYNQK SYYSNSYWYFDL FKG RASSSVSYI H QQWSFNPPT AIYPGNGDTSYNQK SYK SNS D WYFDL FKG RASSSVSYI H QQWSFNPPT AIYPGNG E TSYNQK SYYSNS D WYFDLFKG RASSSVSYI H QQ Y SFNPPT AIYPGNGDTSYNQK SYYSNS D WYFDL FKG RASSSVSYIH QQ Y SFNPPT AIYPGNG E TSYNQK SY K S GGD WYFDL FKG

Mutations were introduced into binding domains of the CD20×CD20 scorpion(2Lm20-4×2Lm20-4) by PCR mutagenesis using primers encoding the alteredsequence region. After sequence confirmation, DNA fragments encoding the2Lm20-4 scFv fragments with corresponding mutations were cloned into aconventional expression vector containing a coding region for theconstant sub-region of a scorpion, resulting in a polynucleotidecontaining the complete DNA sequence of new versions of the2Lm20-4×2Lm20-4 scorpion. The variants of the 2Lm20-4×2Lm20-4 scorpionwith CDR mutations were produced by expression in a transient COS cellsystem and purified through Protein A and size-exclusion (SEC)chromatography. The binding properties of 2Lm20-4×2Lm20-4 scorpionvariants were evaluated by FACS analysis using primary B-cells and theWIL2-S B-lymphoma cell line.

Other mutants have also been generated using a similar approach tooptimize CD20 binding domains. The CD20 SMIP designated TRU015 served asa substrate for generating mutants and, unless noted to the contrary,all domains were human domains. The following mutants were found tocontain useful and functional CD20 binding domains. The 018008 moleculecontained a substitution of Q (single-letter amino acid code) for S atposition 27 of CDR1 in VL, a substitution of S for T at position 28 inCDR1 of VH and a substitution of L for V at position 102 in CDR3 of VH.The following partial scorpion linker sequences, corresponding to theCCCP sequence in an IgG1 hinge, were separately combined with themutated VL and VH: CSCS, SCCS and SCCP, consistent with the modulardesign of scorpions. The 018009 molecule contained a substitution of Qfor S at position 27 of CDR1 of VL, a substitution of S for T atposition 28 of CDR1 of VH and substitutions of S for V at position 96, Lfor V at position 102 and deletion of the V at position 95, all in CDR3of VH. The same scorpion linkers sub-sequences described above as beingfound in the scorpion linkers used in 018008 were used in 018009. The018010 molecule contained substitutions of a Q for S at position 27, anI for M at position 33 and a V for H at position 34, all in CDR1 of VL,along with an S for T substitution at position 28 of CDR1 of VH and an Lfor V substitution at position 102 in CDR3 of VH. Scorpion linkersdefined by the CSCS and SCCS sub-sequences were used with 018010. 018011contained the same mutations in CDR1 of VL and in CDR1 of VH asdescribed for 018010, along with deletion of V at position 95,substitution of S for V at position 96 and substitution of L for V atposition 102, all in CDR3 of VH. Scorpion linkers defined by the CSCS,SCCS and SCCP sub-sequences were used in 018011 molecules. The 018014 VLwas an unmutated mouse VL, with a human VH containing the S for T changeat 28 in CDR1 and the L for V change at 102 in CDR3. 018015 alsocontained an unmutated mouse VL along with a human VH containing an Sfor T change at 28 of CDR1 and, in CDR3, a deletion of V at 95,substitution of S for V at 96, and substitution of L for V at 102. The2Lm5 molecule had a Q for S at 27 in CDR1 of VL, an F for Y at 27 and anS for T at 30, both in CDR1 of VH, as well as deletion of the V at 95, Sfor V at 96 and L for V at 102, all in CDR3 of VH. Scorpion linkersdefined by the CSCS, SCCS and SCCP were separately used in each of018014 and 018015. 2Lm5-1 was the same as 2Lm5 except 2Lm5-1 had nomutations in CDR1 of VH, and only a scorpion linker defined by the CSSSsub-sequence was used. 2Lm6-1 had the mutations of 2Lm5 and asubstitution of T for S at 92 and S for F at 93 in CDR3 of VL, and onlythe scorpion linker defined by the CSSS sub-sequence was used. The onlymutations in 2Lm16 were the mutations in CDR3 of VH listed above for2Lm5-1. Scorpion linkers defined by the sub-sequences CSCS, SCCS, andSCCP were separately used in 2Lm16. 2Lm16-1 substituted Q for S at 27 inCDR1 of VL and substituted T for S at 92, and S for F at 93, both inCDR3 of VL, and, in CDR3 of VH, deleted V at 95, substituted S for V at96 and substituted L for V at 102; only the scorpion linker defined bythe CSSS sub-sequence was used. 2Lm19-3 substituted Q for S at 27, 1 forM at 33, and V for H at 34, all in CDR1 of VL, along with the mutationsin CDR3 of VH listed for 2Lm16-1. Scorpion linkers defined by thesub-sequences CSCS, SCCS, and SCCP were separately used in 2Lm19-3. The2Lm20-4 molecule contained an I for M at 33 and a V for H at 34, both inCDR1 of VL, along with the mutations in CDR3 of VH listed for 2Lm16-1.For 2Lm5-1, 2Lm6-1, 2Lm16, 2Lm16-1, 2Lm19-3, and 2Lm20-4, there also wasan S for L substitution at position 11 in the framework region of VH.Scorpion linkers defined by the CSCS, SCCS and SCCP sub-sequences wereseparately used in 2Lm20-4. Finally, the substitution of S for P atposition 331 was present in the following mutants: 018008 with thescorpion linker defined by CSCS, 018009 with each of scorpion linkersdefined by CSCS and SCCP, 018010 with the scorpion linker defined byCSCS, 018011 with the scorpion linker defined by SCCP, 018014 with thescorpion linker defined by CSCS, 018015 with the scorpion linker definedby CSCS, 2Lm16 with scorpion linkers defined by any of CSCS, SCCS, andSCCP, 2Lm19-3 with a scorpion linker defined by CSCS or SCCP, and2Lm20-4 with a scorpion linker defined by CSCS or SCCP.

In addition, changes in the length of a linker joining two regions of abinding domain, such as regions of a scorpion binding domain thatcorrespond to an Ig V_(L) and V_(H), are contemplated. For example,removal of a C-terminal Asp in interdomain linkers where it is found isexpected to affect the binding characteristics of a scorpion, as is asubstitution of Gly for Asp.

Also contemplated are scorpions that have a scorpion linker (interposedC-terminal to the constant sub-region and N-terminal to binding domain2) that is lengthened relative to a hinge region of an Ig, with aminoacid residues being added C-terminal to any cysteine in the scorpionthat corresponds to an Ig hinge cysteine, with the scorpion cysteinebeing capable of forming an interchain disulfide bond. Scorpionscontaining these features have been constructed and are characterizedbelow.

Efforts were undertaken to improve the expression, stability andtherapeutic potency of scorpions through the optimization of thescorpion linker covalently joining the constant sub-region and theC-terminally disposed binding domain 2. The prototypical scorpion usedfor optimization studies contained an anti-CD20 scFV (binding domain 1)fused N-terminal to the constant sub-region derived from IgG1 C_(H2) andC_(H3), with a second anti-CD20 scFv fused C-terminal to that constantsub-region. This scorpion, like immunoglobulin molecules, is expected toassociate through the constant region (or sub-region) to form ahomodimeric complex with peptide chains linked by disulfide bonds. Toobtain high level of expression of a stable, tetravalent molecule withhigh affinity for its CD20 target, the scorpion linker between theconstant sub-region and the second binding domain must accommodate thefollowing considerations. First, steric hindrance between the homologousbinding domains carried by the two scFv fragments (one scFv fragment oneach of two scorpion monomers) should be minimized to facilitatemaintenance of the native conformations of each binding domain. Second,the configurations and orientations of binding domains should allowproductive association of domains and high-affinity binding of eachbinding domain to its target. Third, the scorpion linker itself shouldbe relatively protease-resistant and non-immunogenic.

In the exemplary CD20×CD20 scorpion construct S0129, the C-terminus ofC_(H3) and the second anti-CD20 scFV domain were linked by the 2H7scorpion linker, a peptide derived from, and corresponding to, afragment of a natural human hinge sequence of IgG1. The 2H7 scorpionlinker served as a base for design efforts using computer-assistedmodeling that were aimed at improving the expression of scorpions andimproving the binding characteristics of the expressed molecules.

To analyze the 2H7 scorpion linker, the 3-dimensional structure of adimeric form of the human IgG1 hinge was modeled using Insight IIsoftware. The crystal structure of anti-CD20 scFV in the V_(H)-V_(L)orientation was chosen as a reference structure for the 20-4 bindingdomains (RCSB Protein Data Bank entry code: 1A14). In intact IgG1, thehinge connects the C-terminus of the C_(H1) domain to the N-terminus ofthe C_(H2) domain, with the configuration of each domain being such thathinge cysteine residues can pair to form a homodimer. In the exemplaryscorpion molecule, the hinge-derived 2H7 linker connected the C-terminalend of the scorpion domain derived from the IgG1 C_(H3) domain to theN-terminal end of that portion of scorpion binding domain 2 derived froman IgG1 V_(H2) domain. Using a 3-D modeled structure of the V_(H)-V_(L)scFV, expectations of the optimal distance between the C-terminal endsof the 2H7 linkers was influenced by three considerations. First, hingestability must be maintained, and stability is aided by dimerization,e.g., homodimerization, which means that the hinge cysteines must beable to pair in the presence of the two folded binding domains. Second,two binding domains, e.g., scFVs, must accommodate the 2H7 linkerC-termini without steric interference in order to allow for properprotein folding. Third, the CDRs of each binding domain should be ableto face the same direction, as in a native antibody, because eachbinding domain of the prototypical scorpion can bind adjacent receptors(CD20) on the same cell surface. Given these considerations, thedistance between the two N-terminal ends of scFvs is expected to beapproximately 28 Å. The distance between the C-terminal ends of thetheoretically designed 2H7 linkers in dimeric scorpion forms is expectedto be about 16 Å. To accommodate the distances expected to be needed foroptimizing the performance of a scorpion, the C-terminus of the 2H7linker was extended by at least 3 amino acids. Such an extension isexpected to allow for the formation of disulfide bonds between 2H7linker cysteine residues, to allow for proper folding of the C-terminalbinding domain 2, and to facilitate a correct orientation of the CDRs.In addition, in intact IgG1, due to the presence of the C_(H1) andV_(L1) domains between the hinge and binding domains, the distancebetween the binding domains carried by the two chains is furtherincreased and is expected to further favor the cross-linking of adjacentreceptors on the same cell surface. In view of the considerationsdescribed above, a set of linkers with different lengths was designed(Table 10). To minimize immunogenicity, natural residues present at theN-terminal end of the C_(H2) domain (Ala-Pro-Glu-Leu or APEL) were usedto lengthen the 2H7 scorpion linker by sequence addition to theC-terminus of the scorpion linker. The longer constructs contained oneor multiple (Gly4Ser) linker units known to be protease-resistant andflexible.

The CD20×CD20 scorpion constructs containing extended scorpion linkersbetween the C_(H3) domain of the constant sub-region and the C-terminalscFv binding domain were constructed using PCR mutagenesis and subclonedinto a conventional mammalian expression vector. The effect of linkerlength on CD20×CD20 scorpion expression could be analyzed be comparingthe yield of secreted protein in transient expression experiments usingCOS or HEK293 cells, or by analysis of protein synthesis andaccumulation in the cells by Western blot analyses or pulse-chasestudies with [35]S-labeled methionine/cysteine.

TABLE 10 Scorpion linker core Extended Construct (2H7) Extensionscorpion linker Number sequence sequence sequence 1 GCPPCPNS APELGCPPCPNSAPEL 2 GCPPCPNS APELGGGGS GCPPCPNSAPELGGGGS 3 GCPPCPNSAPELGGGGSG GCPPCPNS GGGS APELGGGGSGGGGS 4 GCPPCPNS APELGGGGSGG GCPPCPNSGGSGGGGS APELGGGGSGGG GSGGGGS

Glycosylated scorpions are also contemplated and, in this context, it iscontemplated that host cells expressing a scorpion may be cultured inthe presence of a carbohydrate modifier, which is defined herein as asmall organic compound, preferably of molecular weight less than 1000daltons, that inhibits the activity of an enzyme involved in theaddition, removal, or modification of sugars that are part of acarbohydrate attached to a polypeptide, such as occurs during N-linkedcarbohydrate maturation of a protein. Glycosylation is a complex processthat takes place in the endoplasmic reticulum (“core glycosylation”) andin the Golgi bodies (“terminal glycosylation”). A variety of glycosidaseand/or mannosidase inhibitors provide one or more of desired effects ofincreasing ADCC activity, increasing Fc receptor binding, and alteringglycosylation pattern. Exemplary inhibitors include, but are not limitedto, castanospermine and kifunensine. The effects of expressing scorpionsin the presence of at least one such inhibitor are disclosed in thefollowing example.

Example 13 Scorpion Protein Expression Levels and Characterization

Scorpion protein expression levels were determined and the expressedproteins were characterized to demonstrate that the protein design ledto products having practical benefits. A monospecific CD20×CD20 scorpionand a bispecific CD20×CD37 scorpion were expressed in CHO DG44 cells inculture using conventional techniques.

Basal level, stable expression of the CD20×CD20 scorpion S0129(21m20-4×21 m20-4) in CHO DG44 cells cultured in the presence of variousfeed supplements was observed as shown in FIG. 34. All culture mediacontained 50 nM methotrexate, a concentration that maintained copynumber of the scorpion-encoding polynucleotide. The polynucleotidecontained a coding region for the scorpion protein that was notcodon-optimized for expression in CHO DG44 cells. The polynucleotide wasintroduced into cells using the pD18 vector Apparent from FIG. 34,expression levels of about 7-46 μg/ml were obtained.

Expression levels following amplification of the polynucleotide encodinga bispecific CD20×CD37 scorpion were also determined. The pD18 vectorwas used to clone the CD20×CD37 scorpion coding region and the plasmidwas introduced into CHO DG44 cells. Amplification of the encodingpolynucleotide was achieved using the dhfr-methotrexate technique knownin the art, where increasing concentrations of MTX are used to selectfor increased copy number of the Dihydrofolate Reductase gene (dhfr),which leads to co-amplification of the tightly linked polynucleotide ofinterest. FIG. 35 shows that stable expression levels of about 22-118μg/ml of the bispecific CD20×CD37 scorpion were typically observed.Variability in yield was seen under different conditions, includingmethotrexate concentration used for amplification, but these variablesare amenable to optimization by those of skill in the art. A variety ofother scorpion molecules described herein were also subjected toexpression analyses in CHO and/or COS cells, with the results providedin Table 11, below. These results demonstrate that significant yields ofscorpion proteins can be obtained using conventional techniques androutine optimization of the amplification technique.

Expressed proteins were also characterized by SDS-PAGE analysis toassess the degrees of homogeneity and integrity of the expressedproteins and to confirm molecular weight of monomeric peptides. Thedenaturing polyacrylamide gels (4-20% Tris Glycine) were run underreducing and non-reducing conditions. The results presented in FIG. 36reveal single protein bands for each of a 2Lm20-4 SCC SMIP and S1000(CD20(21m20-4)×CD20(21m20-4) monospecific scorpion. S0126) of theexpected monomeric molecular weights under reducing conditions. Thesedata establish that SMIPs and scorpions are amenable to purification inan intact form. Under non-reducing conditions, a trace amount of apeptide consistent with the expected size of a monomeric SMIP was seen,with the vast majority of the protein appearing in a single well-definedband consistent with a dimeric structure. Under these non-reducingconditions, the monospecific scorpion protein showed a singlewell-defined band of a molecular weight consistent with a dimericstructure. The dimeric structures for both the SMIP and the scorpion areconsistent with their monomeric structures, each of which contains ahinge-like scorpion linker containing at least one Cysteine capable ofparticipating in disulfide bond formation.

The effect of scorpion linkers on the expression and integrity ofscorpions was also assessed, and results are shown in Table 12. Thistable lists scorpion linker variants of the monospecific CD20×CD20(2Lm20-4×2Lm20-4) S0129 scorpion and the CD20×CD28 S0033 scorpion(2H7sccpIgG1-H7-2e12), their integrity as single chain molecules, andtheir transient expression levels in COS cells relative to the parentscorpion S0129 or S0033, as appropriate, with an H7 linker (set as100%). Table 13 provides data resulting from an evaluation of scorpionlinker variants incorporated into the CD20×CD20 scorpion, along withanalogous data for the CD20×CD28 scorpion. Table 13 provides dataresulting from an evaluation of S0129 variants containing scorpionlinkers that are not hinge-like linkers containing at least one Cysteinecapable of disulfide bond formation; rather, the scorpion linkers inthese molecules are derived from Type II C-lectin stalks. Apparent fromthe data presented in Table 13 is that hinge-like scorpion linkers maybe associated with scorpions expressed at higher or lower levels than anunmodified parent scorpion linker in transient expression assays.Further, some of the linker variants exhibit greater resistance toproteolytic cleavage than the unmodified parent linker, a concern forall or almost all expressed proteins. The data of Table 13 show thatnon-hinge-like linkers such as linkers derived from the stalk region ofType II C-lectins are found in scorpions that exhibit bindingcharacteristics that vary slightly from scorpions containing hinge-likescorpion linkers. Additionally, the scorpion containing a non-hinge-likescorpion linker exhibits effector function (ADCC) that either equals orexceeds the ADCC associated with scorpions having hinge-like scorpionlinkers.

TABLE 11 Linker Upstream (CH3) S0129 (2Lm20-4 × 2Lm20-4) ExpressionExpression Name Sequence linker variants - aa seq based on #AAS COS¹Cleavage³ CHO² H7 QKSLSLSPGK GCPPCPNS H7 18 100 − 100 H16 QKSLSLSPGKLSVKADFLTPSIGNS CD80 25 174 + H18 QKSLSLSPGK LSVLANFSQPEIGNS CD86 25 165++ H19 QKSLSLSPGK LSVKADFLTPSISCPPCPNS CD86 + H7 30 161 + 109 H25QKSLSLSPGK RIHQMNSELSVLANS CD86 25 170 ++ H32 QKSLSLSPGKRIHLNVSERPFPPCPPCPNS CD22 26 184 ++ H47 QKSLSLSPG LSVKADFLTPSIGNS H16 24141 − 206 H48 QKSLSLSPG KADFLTPSIGNS H16 21 137 − H50 Q LSVLANFSQPEIGNSH18 18 21 − H51 QKS LSVLANFSQPEIGNS H18 18 110 − H52 QKSLSLSPGSQPEIVPISNS H18 20 95 − H53 QXSLSL SQPEIVPISCPPCPNS H19 26 65 − H54 QSVLANFSQPEISCPPCPNS H19 21 72 +/− H55 QKSLSLSPG RIHQMNSELSVLANS H26 24116 + H56 QKSLSLSPG QMNSELSVLANS H26 21 130 − 163 H57 QKSLSLSPGVSERPFPPNS H32 18 118 − H58 QKSLSLSPG KPFFTCGSADTCPNS CD72 24 103 − H59QKSLS KPFFTCGSADTCPNS CD72 23 94 − ¹NFS is a glycosylasion consensusmotif ²Transient expression in COS (6W plates), or CHO (single flask)relative to S0129-H7 (%) ³Cleavage product(s) observed bySDS-PAGE/silver stain. − = none, + = faint band, ++ = major band(s), +++= >50% cleaved

TABLE 12 Linker Linker S0129 (2Lm20-4 × 2Lm20-4) Changes seq. 20 × 2020 × 20 Name linker variants - aa seq in CH3?¹ based on Expression²Cleavage?³ H7 GCPPCPNS N H7 100 − H8 GSPPSPNS N H7 107 + H9 GSPPSPNS YH7 142 − H10 EPKSTDKTHTCPPCPNS N IgG1  98 − hinge H11 EPKSTDKTHTSPPSPNSN IgG1 126 + hinge H16 LSVKADFLTPSIGNS N CD80 174 + H17LSVKADFLTPSISCPPCPNS N CD80 + 113 + H7 H18 LSVLANFSQPEIGNS N CD86 165 ++H19 LSVLANFSQPEISCPPCPNS N CD86 + 161 + H7 H20 LKIQERVSKPKISNS N CD2 115+++ H21 LKIQERVSKPKISCPPCPNS N CD2 + H7  90 +++ H22 LNVSERPFPPHIQNS NCD22 149 ++ H23 LDVSERPFPPHIQSCPPCPNS N CD22 + 121 ++ H7 H24REQLAEVTLSLKANS N CD80 145 ++ H25 REQLAEVTLSLKACPPCPNS N CD80 +  98 + H7H26 RIHQMNSELSVLANS N CD86 170 ++ H27 RIHQMNSELSVLACPPCPNS N CD86 + 154++ H7 H28 DTKGKNVLEKIFSNS N CD2 153 + H30 LPPETQESQEVTLNS N CD22  78 +H32 RIHLNVSERPFPPNS N CD22 184 ++ H33 RIHLNVSERPFPPCPPCPNS N CD22 + 74 + H7 H36 GCPPCPGGGGSNS N H7 110 + H40 GCPPCPANS Y H7 110 + H41GCPPCPANS Y H7 102 − H42 GCPPCPNS Y H7  99 − H44 GGGASCPPCPGNS Y H7108 + H45 GGGASCPPCAGNS Y H7 107 − H46 GGGASCPPCANS Y H7  98 − H47LSVKADFLTPSIGNS Y CD80 141 − H48 ADFLTPSIGNS N CD80 137 − H50LSVLANFSQPEIGNS Y CD86  21 − H51 LSVLANFSQPEIGNS Y CD86 110 − H52SQPEIVPISNS Y CD86  95 − H53 SQPEIVPISCPPCPNS Y CD86 +  95 − H7 H54SVLANFSQPEISCPPCPNS Y CD86 +  72 +/- H7 H55 RIHQMNSELSVLANS Y CD86 118 +H56 QMNSELSVLANS Y CD86 130 − H57 VSERPFPPNS Y CD22 118 − H58KPFFTCGSADTCPNS Y CD72 103 − H59 KPFFTCGSADTCPNS Y CD72  94 − H60QYNCPGQYTFSMNS Y CD69 >100⁵ − H61 EPAFTPGPNIELQKDSDCNS Y CD94 >100  −H62 QRHNNSSLNTRTQKARHCNS Y NKG2A >100  − H63 NSLFNQEVQIPLTESYCNS YNKG2D >100  − ¹Additional changes to the end of CH3 such as 1-9 aadeletion and/or codon optimization ²Transient expression in COS (6Wplates), relative to S0129-H7 parent (%) ³Cleavage product(s) observedby SDS-PAGE/silver stain: − = none, + = faint band, ++ = major band(s),+++ >50% cleaved ⁵H60-H63 variants compared by estimation of recovery ofprotein purified from COS spent media.

TABLE 13 Production Yield % POI (ug (M.wt Improvement protein in Kd overpurified/ml by S0129wt Binding to ADCC Sequence of Proteins Descriptionsup) MALS) POI Ramos assay scorpion linker S0129wt H7 linker 1.6  67 — —— GCPPC (167) S0129- CD69 stalk 2.9  66 1.8 Weaker *Slightly QYNCPGQYTFCD69 (167) than better than SM S0129 wt S0129wt POI S0129- CD72 2.0  691.2 Similar to *Slightly PFFTCGSADTC CD72 truncated (165) S0129wtbetter than stalk S0129wt POI S0129- CD94 stalk 2.9  67 1.8 Similar to*Slightly EPAFTPGPNIE CD94 (171) S0129wt better than LQKDSDC S0129wt POIS0129- NKG2A 2.5  93 2.2 Slightly Similar to QRHNNSSLNT NKG2A stalk(170) better than S0129wt RTQKARHC S0129wt POI S0129- NKG2D 1.9  70 1.2Similar to *Slightly NSLFNQEVQIP NKG2D stalk (166) S0129wt better thanLTESYC S0129wt POI

As noted in the preceding example, production by expression of scorpionsin cultures containing a carbohydrate modifier is contemplated. Inexemplary embodiments, castanospermine (MW 189.21) is added to theculture medium to a final concentration of about 200 μM (correspondingto about 37.8 μg/mL), or concentration ranges greater than about 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 μM, and upto about 300, 275, 250, 225, 200, 175, 150, 125, 100, 75, 60, or 50μg/mL. For example, ranges of 10-50, or 50-200, or 50-300, or 100-300,or 150-250 μM are contemplated. In other exemplary embodiments, DMJ, forexample DMJ-HCl (MW 199.6) is added to the culture medium to a finalconcentration of about 200 μM (corresponding to about 32.6 μg DMJ/mL),or concentration ranges greater than about 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 110, 120, 130, 140, or 150 μM, and up to about 300, 275,250, 225, 200, 175, 150, 125, 100, 75, 60, or 50 mg/mL. For example,ranges of 10-50, or 50-200, or 50-300, or 100-300, or 150-250 μM arecontemplated. In other exemplary embodiments, kifunensine (MW 232.2) isadded to the culture medium to a final concentration of about 10 μM(corresponding to about 2.3 μg/mL), or concentration ranges greater thanabout 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 μM, and up to about 50, 45,40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, or 11 μM. Forexample, ranges of 1-10, or 1-25, or 1-50, or 5-10, or 5-25, or 5-15 μMare contemplated.

In one experiment, a monospecific CD20×CD20 scorpion (S0129) wasexpressed in cells cultured in 200 μM castanospermine (S0129 CS200) or10 μM (excess) kifunensine (S0129 KF 10) and the binding, or staining,of WIL2S cells by the expressed scorpion was measured, as shown in FIG.42. In comparative binding studies, moreover, a glycosylated S0129scorpion bound CD16 (FCγRIII) approximately three times better than theunglycosylated S0129 scorpion.

In another study, the ADCC-mediated killing of BJAB B-cells by humanizedCD20×CD20 scorpion (S0129) was explored. The results shown in FIG. 43establish that the scorpion, when expressed in cells being cultured inthe presence of either castanospermine or kifunensine, led tosignificantly more potent ADCC-mediated BJAB B-cell death for a givenconcentration of scorpion exposure.

Example 14 Scorpion Binding

a. Domain Spacing

Bispecific scorpions are capable of binding at least two targetssimultaneously, utilizing the pairs of binding domains at the N- andC-terminus of the molecule. In so doing, for cell-surface targets, thecomposition can cross-link or cause the physical co-approximation of thetargets. It will be appreciated by those skilled in the art that manyreceptor systems are activated upon such cross-linking, resulting insignal induction causing changes in cellular phenotype. The design ofthe compositions disclosed herein was intended, in part, to maximizesuch signaling and to control the resultant phenotype.

Approximate dimensions of domains of the scorpion compositions, as wellas expectations of interdomain flexibility in terms of ranges ofinterdomain angles, are known and were considered in designing thescorpion architecture. For scorpions using scFv binding domains forbinding domains 1 and 2 (BD1 and BD2), an IgG1 N-terminal hinge (H1),and the H7 PIMS linker described herein, the binding domain at theN-terminus and the binding domain at the C-terminus may be maximallyabout 150-180 Å apart and minimally about 20-30 Å apart. Binding domainsat the N-terminus may be maximally about 90-100 Å apart and minimallyabout 10-20 Å apart (Deisenhofer, et al., 1976, Hoppe-Seyler's Z.Physiol. Chem. Bd. 357, S. 435-445; Gregory, et al., 1987, Mol. Immunol.24(8):821-9; Poljak, et al., 1973, Proc. Natl. Acad. Sci., 1973, 70:3305-3310; Bongini, et al., 2004, Proc. Natl. Acad. Sci. 101: 6466-6471;Kienberger, et al., 2004, EMBO Reports, 5: 579-583, each incorporatedherein by reference). The choice of these dimensions was done in part toallow for receptor-receptor distances of less than about 50 Å inreceptor complexes bound by the scorpion as distances less than this maybe optimal for maximal signaling of certain receptor oligomers (Paar, etal., 2002, J. Immunol., 169: 856-864, incorporated herein by reference)while allowing for the incorporation of F_(C) structures required foreffector function.

The binding domains at the N- and C-terminus of scorpions were designedto be flexible structures to facilitate target binding and to allow fora range of geometries of the bound targets. It will also be appreciatedby those skilled in the art that flexibility between the N- orC-terminal binding domains (BD1 and BD2, respectively) and between thebinding domains and the F_(C) domain of the molecule, as well as themaximal and minimal distances between receptors bound by BD1 and/or BD2,can be modified, for example by choice of N-terminal hinge domain (H1)and, by structural analogy, the more C-terminally located scorpionlinker domain (H2). For example hinge domains from IgG1, IgG2, IgG3,IgG4, IgE, IgA2, synthetic hinges and the hinge-like C_(H2) domain ofIgM show different degrees of flexibility, as well as different lengths.Those skilled in the art will understand that the optimal choice of H1and scorpion linker (H2) will depend upon the receptor system(s) thescorpion is designed to interact with as well as the desired signalingphenotype induced by scorpion binding.

In some embodiments, scorpions have a scorpion linker (H2) that is ahinge-like linker corresponding to an Ig hinge, such as an IgG1 hinge.These embodiments include scorpions having an amino acid sequence of thescorpion hinge that is an N-terminally extended sequence relative to,e.g., the H7 sequence or the wild-type IgG1 hinge sequence. Exemplaryscorpion linkers of this type would have the sequence of the H7 hingeN-terminally extended by H₂N-APEL(x)_(y)-CO₂H, where x is a unit of theGly₄Ser linker and y is a number between 0 and 3. Exemplifying theinfluence of the scorpion linker on scorpion stability is a study doneusing two scorpions, a bispecific CD20×CD28 scorpion and a monospecificCD20×CD20 scorpion. For each of these two scorpion designs, a variety ofscorpion linkers were inserted. In particular, scorpion linkers H16 andH17, which primarily differ in that H17 has the sequence of H16 with thesequence of H7 appended at the C-terminus, and scorpion linkers H18 and19, in which analogously the sequence of H7 is appended at theC-terminus of H18 in generating H19. For each of the two scorpionbackbones (20×28 and 20×20), each of the four above-described scorpionlinkers were inserted at the appropriate location. Transient expressionof these constructs was obtained in COS cells and the scorpion proteinsfound in the culture supernatants were purified on protein A/G-coatedwells (Pierce SEIZE IP kit). Purified proteins were fractionated onSDS-PAGE gels and visualized by silver stain. Inspection of FIG. 44reveals that the additional H7 sequence in the scorpion linker adds tothe stability of each type of scorpion linker and each type of scorpionprotein. In other words, appending H7 to the C-terminus of either H16 orH18 added to the stability of the scorpion molecule, and thisobservation held regardless of whether the scorpion was CD20×CD28 orCD20×CD20. In terms of target binding, the scorpion proteins having theCD20×CD20 architecture exhibited similar binding properties to theparent monospecific humanized CD20×CD20 scorpion S0129, as shown in FIG.45.

Beyond the preceding embodiments, however, it may be desirable toprevent bound receptors from approaching within about 50 Å of each otherto intentionally create submaximal signals (Paar, et al., J. Immunol.,169: 856-864). In such a case, choices of H1 and Scorpion linker (H2)that are shorter and less flexible than those described above would beexpected to be appropriate.

The same spacing considerations apply to scorpion linkers that are nothinge-like. These scorpion linkers are exemplified by the class ofpeptides having the amino acid sequence of a stalk region of a C-lectin.Exemplary scorpion hinges comprising a C-lectin stalk region arescorpion hinges derived from the CD72 stalk region, the CD94 stalkregion, and the NKG2A stalk region. Scorpions containing such scorpionhinges were constructed and characterized in terms of expression,susceptibility to cleavage, and amenability to purification. The dataare presented in Table 14.

TABLE 14 Bench-top Linker G₄S Codon End ofS0129 Scorpion Linker variants Linker seq. Expression purification Nameoptimization¹ CH3 amino acid seq based on (% S0129)² Cleavage³ % POI H7N K GCPPCPNS H7 100 — 70 H60 Y(17) K GCPPCPNS H7 114 — ND H61 Y(15) KGCPPCPNS H7 90 — 66 H62 N G QRHNNSSLNTRTQKARHCPNS NKG2A stalk 129 — 89H63 Y(17) G QRHNNSSLNTRTQKARHCPNS NKG2A stalk 100 — 85 H64 Y(15) GQRHNNSSLNTRTQKARHCPNS NKG2A stalk 81 — 83 H65 N G EPAFTPGPNIELQKDSDCPNSCD94 stalk 133 — 66 H66 Y(17) G EPAFTPGPNIELQKDSDCPNS CD94 stalk 200 —64 H67 Y(15) G EPAFTPGPNIELQKDSDCPNS CD94 stalk 129 — 65 H68 N GRTRYLQVSQQLQQTNRVLEVTNSSLRQQLR CD72 full 110 — 75LKITQLGQSAEDLQGSRRELAQSQEALQVEQ stalk RAHQAAEGQLQACQADRQKTKETLQSEEQQRRALEQKLSNMENRLKPFFTCGSADTC ¹Codon optimization of Gly₄Ser linker, with(17) or without (15) restriction site ²Estimate of expression in COSbased on recovery of protein in benchtop purification ³Cleavageproduct(s) observed by SDS-PAGE/Coomassie Blue stain of purified protein

b. Binding of N- and C-Terminal Binding Domains

Both N- and C-Terminal Domains Participate in Target Cell Binding

The target cell binding abilities of a CD20 SMIP (TRU015), a CD37 SMIP(SMIP016), a combination of CD20 and CD37 SMIPS (TRU015+SMIP016), andthe CD20×CD37 bispecific scorpion (015×016), were assessed by measuringthe capacity of each of these molecules to block the binding of anantibody specifically competing for binding to the relevant target,either CD37 or CD20. The competing antibodies were FITC-labeledmonoclonal anti-CD37 antibody or PE-labeled monoclonal anti-CD20antibody, as appropriate. Ramos B-cells provided the targets.

Ramos B-cells at 1.2×10⁷/ml in PBS with 5% mouse sera (#100-113, GeminiBio-Products, West Sacramento, Calif.) (staining media) were added to96-well V-bottom plates (25 μl/well). The various SMIPs and scorpionswere diluted to 75 μg/ml in staining media and 4-fold dilutions wereperformed to the concentrations indicated in FIG. 38. The dilutedcompounds were added to the plated cells in addition to media alone forcontrol wells. The cells were incubated for 10 minutes with thecompounds and then FITC anti-CD37 antibody (#186-040, Ancell, Bayport,Minn.) at 5 μg/ml and PE anti-CD20 antibody (#555623, BD Pharmingen, SanJose, Calif.) at 3 μg/ml (neet) were added together to the wells in 25μl staining media. The cells were incubated on ice in the dark for 45minutes and then washed 2.5 times with PBS. Cells were fixed with 1%paraformaldehyde (#19943 1 LT, USB Corp, Cleveland, Ohio) and then runon a FACs Calibur (BD Biosciences, San Jose, Calif.). The data wereanalyzed with Cell Quest software (BD Biosciences, San Jose, Calif.).The results shown in FIG. 38 establish that all SMIPs, SMIP combinationsand scorpions containing a CD20 binding site successfully competed withPE-labeled anti-CD 20 antibody for binding to Ramos B-cells (upperpanel); all SMIPs, SMIP combinations and scorpions containing a CD37binding site successfully competed with FITC-labeled anti-CD 37 antibodyfor binding to Ramos B-cells (lower panel). The bispecific CD20×CD37scorpion, therefore, was shown to have operable N- and C-terminalbinding sites for targets on B-cells.

c. Cell-Surface Persistence

An investigation of the cell-surface persistence of bound SMIPs andscorpions (monospecific and bispecific) on the surface of B-cellsrevealed that scorpions exhibited greater cell-surface persistence thanSMIPs. Ramos B-cells at 6×10⁶/ml (3×10⁵/well) in staining media (2.5%goat sera, 2.5% mouse sera in PBS) were added to 96-well V-bottomplates. Test reagents were prepared at two-fold the final concentrationin staining media by making a 5-fold serial dilution of a 500 nM initialstock and then were added 1:1 to the Ramos B-cells. In addition, mediacontrols were also plated. The cells were incubated in the dark, on ice,for 45 minutes. The plates were then washed 3.5 times with cold PBS. Thesecondary reagent, FITC goat anti-human IgG (#H10501, Caltag/Invitrogen,Carlsbad, Calif.) was then added at a 1:100 dilution in staining media.The cells were incubated for 30 minutes in the dark, on ice. Cells werethen washed 2.5 times by centrifugation with cold PBS, fixed with a 1%paraformaldehyde solution (#19943 1 LT, USB Corp, Cleveland, Ohio) andthen run on a FACs Calibur (BD Biosciences, San Jose, Calif.). The datawere analyzed with CellQuest software (BD Biosciences, San Jose,Calif.). Results of the data analysis are presented in FIG. 37, whichshows the binding of several SMIPs, a monospecific CD20×CD20 scorpionand a bispecific CD20×CD37 scorpion to their targets on Ramos B cells.

Two tubes of Ramos B-cells (7×10⁵/ml) were incubated for 30 minutes onice with each of the two compounds being investigated, i.e., a humanizedCD20 (2Lm20-4) SMIP and a humanized CD20×CD20 (2Lm20-4×2Lm20-4)scorpion, each at 25 μg/ml in Iscoves media with 10% FBS. At the end ofthe incubation period, both tubes were washed 3 times by centrifugation.One tube of cells was then plated into 96-well flat-bottom plates at2×10⁵ cells/well in 150 μl of Iscoves media with one plate then goinginto the 37° C. incubator and the other plate incubated on ice. Thesecond tube of each set was resuspended in cold PBS with 2% mouse serumand 1% sodium azide (staining media) and plated into a 96-well V-bottomplate at 2×10⁵ cells/well for immediate staining with the secondaryantibody, i.e., FITC goat anti-human IgG (#H10501, Caltag/Invitrogen,Carlsbad, Calif.). The secondary antibody was added at a 1:100 finaldilution in staining media and the cells were stained on ice, in thedark, for 30 minutes. Cells were then washed 2.5 times with cold PBS,and fixed with 1% paraformaldehyde (#19943 1 LT, USB Corp, Cleveland,Ohio).

At the time points designated in FIG. 39, samples were harvested fromthe 96-well flat-bottom plates, incubated at either 37° C. or on ice,and placed into 96-well V-bottom plates (2×10⁵ cells/well). The cellswere washed once with cold staining media, resuspended, and thesecondary antibody was added at a final dilution of 1:100 in stainingmedia. These cells were incubated on ice, in the dark, for 30 minutes.The cells were then washed 2.5 times by centrifugation in cold PBS, andsubsequently fixed with 1% paraformaldehyde. The samples were run on aFACS Calibur (BD Biosciences, San Jose, Calif.) and the data wasanalyzed with CellQuest software (BD Biosciences, San Jose, Calif.).Results presented in FIG. 39 demonstrate that the binding of a SMIP anda scorpion to the surface of B-cells persists for at least six hours,with the monospecific hu CD20×CD20 (2Lm20-4×2Lm20-4) scorpion persistingto a greater extent than the hu CD20 (2Lm20-4) SMIP.

Example 15 Direct Cell Killing by Monospecific and Bispecific Scorpions

Experiments were conducted to assess the capacity of monospecific andbispecific scorpion molecules to directly kill lymphoma cells, i.e., tokill these cells without involvement of ADCC or CDC. In particular, theSu-DHL-6 and DoHH2 lymphoma cell lines were separately subjected to amonospecific scorpion, i.e., a CD20×CD20 scorpion or a CD37×CD37scorpion, or to a bispecific CD20×CD37 scorpion.

Cultures of Su-DHL-6, DoHH2, Rec-1, and WSU-NHL lymphoma cells wereestablished using conventional techniques and some of these cultureswere then individually exposed to a monospecific CD20 SMIP, amonospecific scorpion (CD20×CD20 or CD37×CD37), or a bispecific scorpion(CD20×CD37 or CD19×CD37). The exposure of cells to SMIPs or scorpionswas conducted under conditions that did not result in cross-linking. Thecells remained in contact with the molecules for 96 hours, after whichgrowth was measured by detection of ATP, as would be known in the art.The cell killing attributable to the CD20 SMIP and the CD20×CD20monospecific scorpion are apparent in FIG. 24 and Table 15. The cellkilling capacity of the CD37×CD37 monospecific scorpion is apparent fromFIG. 25 and Table 15, the ability of the CD20×CD37 bispecific scorpionto kill lymphoma cells is apparent from FIG. 26 and Table 15, and thecapacity of the CD19×CD37 bispecific scorpion to kill lymphoma cells isevident from FIG. 27 and Table 15. Data were pooled from threeindependent experiments and points represent the mean±SEM. IC₅₀ valuesin Table 15 were determined from the curves in FIGS. 24, 25, and 26, asnoted in the legend to Table 15, and are defined as the concentrationresulting in 50% inhibition compared to untreated cultures. The data inthe figures and table demonstrate that scorpions are greater than10-fold more potent in killing these cell lines than the free SMIP usingthe same binding domains.

TABLE 15 Cell Line IC₅₀ (nM) SU-DHL-6 DoHH2 WSU-NHL CD20 SMIP* >100 60NA CD20xCD20 0.3 4.0 NA scorpion* CD37 SMIP** >100 >100 NA CD37xCD37 101.2 NA scorpion** CD20 SMIP and 6 2 NA CD37 SMIP*** CD20xCD37 0.05 0.05NA scorpion*** CD19 SMIP and 0.16 NA 0.40 CD37 SMIP**** CD19xCD37 0.005NA 0.04 scorpion**** *Data derived from FIG. 24. **Data derived fromFIG. 25. ***Data derived from FIG. 26. ****Data derived from FIG. 27.

Additional experiments with the humanized CD20×CD20 scorpion S0129 wereconducted in Su-DHL-4, Su-DHL-6, DoHH2, Rec-1, and WSU-NHL cells. Theresults are presented in FIG. 46 and FIG. 47. The data provided in thesefigures extends the findings discussed above in showing that scorpionshave the capacity to directly kill a variety of cell lines.

The above findings were extended to other monospecific and bispecificscorpions, with each scorpion demonstrating capacity to directly kill Bcells. DoHH2 B-cells were exposed in vitro to the monospecific CD20×CD20scorpion, a monospecific CD37×CD37 scorpion, or a bispecific CD20×CD37scorpion. The results presented in FIG. 48 demonstrate that bispecificscorpions have kill curves that are different in form from monospecificscorpions.

Culturing Su-DHL-6 cells in the presence of 70 nM CD20×CD20 scorpion(S0129), CD20×CD37 scorpion, or CD37×CD37 scorpion also led to directB-cell killing in an in vitro environment (FIG. 49). Consistently,Su-DHL-6 cells exposed to either a bispecific CD19×CD37 scorpion or toRituxan® led to direct cell killing, with the bispecific scorpionexhibiting lethality at lower doses, as revealed in FIG. 50.

Another demonstration of direct cell killing was provided by exposingDHL-4 cells to four independent monospecific scorpions recognizing CD20.Two versions of CD20×CD20 scorpion were designed to incorporate two 20-4binding domains (20-4×20-4 and S0129) and the second two incorporate ahybrid of the 011 and 20-4 binding domains. All four of theindependently constructed and purified versions of the two CD20×CD20scorpion designs, (20-4×20-4 and S0129) and hybrid (011×20-4 and011×20-4ΔAsp), efficiently killed the DHL-4 cells in a direct manner.For this study, DHL-4 cells were treated in vitro with 1 μg/ml of theindicated proteins for 24 hours. Cells were then stained with Annexin Vand Propidium Iodide, early and late markers of cell death,respectively, and cell populations were quantified by FACS. The resultspresented in FIG. 51 establish the direct killing capacity of each ofthe CD20×CD20 constructs as evidenced by increased staining shown inblack bars. In addition, the results demonstrate that the hybrid011×20-4 proteins exhibited a slight increase in direct cell killingrelative to 20-4×20-4-based scorpions, despite the fact that each ofthese scorpions monospecifically recognized CD20. In a separate set ofexperiments, the dose-response of the four independent scorpionconstructs was determined by FACS analysis of Annexin V- and PropidiumIodide-stained cell populations. The results, shown in FIG. 52,demonstrate dose-responsive increases in cell death resulting fromtreatment of the DHL-4 cells with each of the independent scorpionconstructs.

Example 16 Accessory Functions Mediated by Scorpions (ADCC & CDC)

a. Scorpion-Dependent Cellular Cytotoxicity

Experiments were conducted to determine whether scorpions would mediatethe killing of BJAB B lymphoma cells. BJAB B lymphoma cells wereobserved to be killed with CD20 and/or CD37 scorpions.

Initially, 1×10⁷/ml BJAB B-cells were labeled with 500 μCi/ml ⁵¹Crsodium chromate (#CJS1, Amersham Biosciences, Piscataway, N.J.) for 2hours at 37° C. in Iscoves media with 10% FBS. The ⁵¹Cr-loaded BJAB Bcells were then washed 3 times in RPMI media with 10% FBS andresuspended at 4×10⁵/ml in RPMI. Peripheral blood mononuclear cells(PBMC) from in-house donors were isolated from heparinized whole bloodvia centrifugation over Lymphocyte Separation Medium (#50494, MPBiomedicals, Aurora, Oh), washed 2 times with RPMI media and resuspendedat 5×10⁶/ml in RPMI with 10% FBS. Reagent samples were added to RPMImedia with 10% FBS at 4 times the final concentration and three 10-foldserial dilutions for each reagent were prepared. These reagents werethen added to 96-well U-bottom plates at 50 μl/well to the indicatedfinal concentrations. The ⁵¹Cr-labeled BJAB were then added to theplates at 50 μl/well (2×10⁴/well). The PBMC were then added to theplates at 100 μl/well (5×10⁵/well) for a final ratio of 25:1 effectors(PBMC):target (BJAB). Effectors and targets were added to media alone tomeasure background killing. The ⁵¹Cr-labeled BJAB were added to mediaalone to measure spontaneous release of ⁵¹Cr and to media with 5% NP40(#28324, Pierce, Rockford, Ill.) to measure maximal release of ⁵¹Cr. Theplates were incubated for 6 hours at 37° C. in 5% CO₂. Fifty μl (25 μlwould also be suitable) of the supernatant from each well were thentransferred to a LumaPlate-96 (#6006633, Perkin Elmer, Boston, Mass.)and dried overnight at room temperature.

After drying, radioactive emissions were quantitated as cpm on a PackardTopCount-NXT. Sample values were the mean of triplicate samples. Percentspecific killing was calculated using the following equation: %Kill=((sample−spontaneous release)/(maximal release−spontaneousrelease))×100. The plots in FIG. 30 show that BJAB B cells were killedby monospecific scorpions CD20×CD20 and CD37×CD37. The combination ofCD20 SMIP and CD37 SMIP also killed BJAB B cells. These resultsdemonstrate that scorpions exhibit scorpion-dependent cellularcytotoxicity and it is expected that this functionality is provided bythe constant sub-region of the scorpion, providing ADCC activity.

b. Scorpion Role in Complement-Dependent Cytotoxicity

Experiments also demonstrated that scorpions have Complement-DependentCytotoxicity (CDC) activity. The experiment involved exposure of RamosB-cells to CD19 and/or CD37 SMIPs and scorpions, as described below andas shown in FIG. 31.

The experiment was initiated by adding from 5 to 2.5×10⁵ Ramos B-cellsto wells of 96-well V-bottomed plates in 50 μl of Iscoves media (noFBS). The test compounds in Iscoves, (or Iscoves alone) were added tothe wells in 50 μl at twice the indicated final concentration. The cellsand reagents were incubated for 45 minutes at 37° C. The cells werewashed 2.5 times in Iscoves with no FBS and resuspended in Iscoves withhuman serum (# A113, Quidel, San Diego, Calif.) in 96-well plates at theindicated concentrations. The cells were then incubated for 90 minutesat 37° C. The cells were washed by centrifugation and resuspended in 125μl cold PBS. Cells were then transferred to FACs cluster tubes (#4410,CoStar, Corning, N.Y.) and 125 μl PBS with propidium iodide (# P-16063,Molecular Probes, Eugene, Oreg.) at 5 μg/ml was added. The cells wereincubated with the propidium iodide for 15 minutes at room temperaturein the dark and then placed on ice, quantitated, and analyzed on aFACsCalibur with CellQuest software (Becton Dickinson). The resultspresented in FIG. 31 establish that the CD19 SMIP, but not the CD37SMIP, exhibits CDC activity, with a combination of the two SMIPsexhibiting approximately the same level of CDC activity as CD19 SMIPalone. The CD19×CD37 scorpion, however, exhibited significantly greaterCDC activity than either SMIP alone or in combination, establishing thatthe scorpion architecture provides a greater level ofComplement-dependent Cytotoxicity than other molecular designs.

c. ADCC/CDC Activity of CD20×CD20 Monospecific Scorpions

Three distinct CD20×CD20 monospecific scorpions were examined for ADCCand CDC functionality, along with appropriate controls. ADCC was assayedusing conventional techniques, and the results are presented in FIG. 53.Apparent from the Figure is the appreciable, but not identical, ADCCactivity associated with each of the tested CD20×CD20 monospecificscorpions.

To assess CDC, Ramos B-cell samples (4×10⁵) were incubated with each ofthe CD20×CD20 scorpions (0, 0.5, 5, 50 and 500 nM) and scrum (10%) for3.5 hour at 37° C. Cell death was assessed by 7-AAD staining and FACSanalysis. The results are presented in FIG. 54, which reveals that thescorpions exhibit some CDC activity. In a similar experiment, RamosB-cell samples (4×10⁵) were incubated with CD20×CD20 scorpion protein(5, 50, 100 nM) and serum (10%) for 2 hour at 37° C. Cells were washed2× and incubated with anti-human C1q FITC antibody. Bound C1q wasassessed by FACS analysis and the results are presented in FIG. 55.These results are consistent with the results presented in FIG. 54 thateach of the CD20×CD20 monospecific scorpions was associated with someCDC activity, although less activity than was associated with a CD20SMIP.

d. Interactions of Scorpions with F_(C)γRIII

ELISA studies showed that scorpions bound to FcγRIII (CD16) low (a lowaffinity isoform or allelotype) at increased levels in the absence oftarget cells. ELISA plates were initially coated with either low- orhigh-affinity CD16mIgG using conventional techniques. The ability ofthis immobilized fusion protein to capture either a CD20 SMIP or aCD20×CD20 monospecific scorpion was assessed. Bound SMIPs and scorpionswere detected with goat anti-human IgG (HRP) secondary antibody and meanfluorescence intensity (MFI) was determined. PBS alone (negativecontrol) is shown as a single point. The results are presented in FIG.32A (capture by CD16 high affinity isoform fusion) and 32B (capture byCD16 low affinity isoform fusion). Apparent from a consideration ofFIGS. 32A and 32B is that both CD20 SMIP and CD20×CD20 monospecificscorpion showed increased binding to both the high- and low-affinityCD16 isoform fusions, with the CD20×CD20 scorpion showing a dramaticincrease in binding to the low affinity isoform fusion with increasingprotein concentration.

The binding of scorpions to the FcγRIII isoforms in the presence oftarget cells was also assessed. The data show the increased binding ofscorpions to both FcγRIII (CD16) low- and high-affinity isoforms orallelotypes in the presence of target cells with increasing proteinconcentration.

In conducting the experiment, CD20-positive target cells were exposed toCD20 SMIPs or CD20×CD20 monospecific scorpions under conditions thatallowed the binding of the SMIP or scorpion to the CD20-positive targetcell. Subsequently, the SMIP- or scorpion-bearing target cell wasexposed to either CD16 high- or low-affinity isoform tagged with mouseIgFc. A labeled goat anti-mouse Fc was then added as a secondaryantibody to label the immobilized CD16 tagged with the mouse IgFc. Cellswere then detected using flow cytometry on a FACs Calibur (BDBiosciences, San Jose, Calif.) and analyzed with Cell Quest software (BDBiosciences, San Jose, Calif.). As shown in FIG. 33, increasedconcentrations of each of the CD20 SMIP and the CD20×CD20 monospecificscorpion led to increased binding to the CD16 isoforms in the presenceof target cells, with the increase in binding of the CD20×CD20 scorpionbeing more significant than the increased binding seen with the CD20SMIP.

Example 17 Cell-Cycle Effects of Scorpions on Target Lymphoma Cells

The cell-cycle effects of scorpions were assessed by exposing lymphomacells to SMIPs, monospecific scorpions and bispecific scorpions. Moreparticularly, DoHH2 lymphoma cells (0.5×10⁶) were treated for 24 hourswith 0.4 nM rituximab, CD20×CD37 scorpion, TRU-015 (CD20 SMIP)+SMIP-016combination (0.2 nM each), 100 nM SMIP-016 or 100 nM CD37×CD37 scorpion.These concentrations represent about 10-fold more than the IC50 value ofthe scorpion in a 96-hour growth inhibition assay (see FIGS. 24-27).Cultures were labeled for 20 minutes at 37° C. with 10 μM BrdU(bromodeoxyuridine). Following fixation, cells were stained withanti-BrdU-FITC antibody and counterstained with propidium iodide. Valuesin FIG. 28 are the mean+/−SD of 4 replicate cultures from 2-3independent experiments. All sample data were analyzed at the same timeand pooled for presentation using both the BrdU and PI incorporation dotplots. Plots demonstrate that a major effect of scorpion treatment is adepletion of cells in S-phase, as well as an increase in the G₀/G₁compartment.

Example 18 Physiological Effects of Scorpions

a. Mitochondrial Potential

CD20×CD20 scorpions induced loss of mitochondrial membrane potential inDHL4 B-cells, as revealed in a JC-1 assay. JC-1 is a cationiccarbocyanine dye that exhibits potential-dependent accumulation in themitochondria (Mitoprobe® JC-1 Assay Kit for Flow Cytometry fromMolecular Probes). JC-1 is more specific to the mitochondrial membranethan the plasma membrane and is used to determine changes inmitochondrial membrane potential. Accumulation in mitochondria isindicated by a fluorescence shift from green (529 nm) to red (590 nm).

In conducting the experiment, DHL-4 B-cells (5×10⁵ cells/ml) wereinitially cultured in 24-well plates and treated for 24 hours with 1μg/ml CD20×CD20 scorpion, Rituximab, IgG control antibody, or 5 μMstaurosporine at 37° C., 5% CO₂, in a standard tissue-culture incubator.JC-1 dye (10 μl/ml, 2 μM final concentration) was added and cells wereincubated for another 30 minutes at 37° C. Cells were harvested bycentrifugation (5 minutes at 1200 rpm), washed with 1 ml PBS, andresuspended in 500 μl PBS. Cells were analyzed by flow cytometry(FACSCalibur, BD) with 488 nM excitation and 530 nM and 585 nM emissionfilters. For the representative scatter plots shown in FIG. 56, redfluorescence was measured on the Y-axis and green fluorescence wasmeasured on the X-axis. Depolarization of the mitochondrial membrane wasmeasured as a decrease in red fluorescence, as seen in the positivecontrol CCCP (carbonyl cyanide 3-chlorophenylhydrazone), a knownmitochondrial membrane potential disrupter. To confirm that JC-1 wasresponsive to changes in membrane potential, DHL-4 B-cells were treatedwith two concentrations of CCCP (50 μM and 250 μM) for 5 minutes at 37°C., 5% CO₂. An additional positive control was cells treated with thebroad-spectrum kinase inhibitor staurosporine to induce apoptosis. Theresults shown in FIG. 56 are dot-plot graphs of 10,000 counts, with redfluorescence plotted on the Y-axis and green fluorescence plotted on theX-axis. A summary histogram of the percentage of cells with disruptedmitochondrial membrane potential (disrupted MMP: black bars) is shown inFIG. 56. These results demonstrate that treatment with either the20-4×20-4 scorpion or the 011×20-4 scorpion generated a decrease in themitochondrial membrane potential associated with cell death.

b. Calcium Flux

Scorpion molecules were analyzed for influences on cell signalingpathways, using Ca⁺⁺ mobilization, a common feature of cell signaling,as a measure therefor. SU-DHL-6 lymphoma cells were labeled with Calcium4 dye and treated with the test molecules identified below. Cells wereread for 20 seconds to determine background fluorescence, and thenSMIPs/scorpions were added (first dashed line in FIG. 28) andfluorescence was measured out to 600 seconds. At 600 seconds, an 8-foldexcess of cross-linked goat-anti-human F(ab)′2 was added andfluorescence was measured for a further 300 seconds. Panel (A) of FIG.28 shows the results obtained with a combination of CD20 SMIP and CD37SMIP (red line); or obtained with a CD20×CD37 bispecific scorpion (blackline), compared with unstimulated cells (blue line). In panel B of FIG.28, the results of treating cells with CD20 SMIP alone (red line)resulted in Ca⁺⁺ mobilization, but this was not as robust as the signalgenerated by the monospecific CD20×CD20 scorpion (black line). The Ca⁺⁺mobilization plots of FIG. 28 represent the fluorescence from triplicatewells treated with equimolar amounts of scorpion and SMIP/SMIPcombinations.

c. Caspases 3, 7 and 9

The ability of CD20-binding scorpions to directly kill B-cells asevidenced by increased Annexin V and Propidium Iodide staining and theloss of mitochondrial membrane potential led to an further investigationof additional apoptosis-related effects of CD20-binding scorpions inB-cells. The approach taken was to perform Apo1 assays on DHL-4 B-cellsexposed to CD20×CD20 scorpions or appropriate controls. The Apo1 assayis based on a synthetic peptide substrate for caspase 3 and 7. The assaycomponents are available from Promega (Apo-ONE® Homogeneuous Caspase-3/7Assay). Caspase-mediated cleavage of the labeled peptideZ-DEVD-Rhodamine 110 releases the fluorescent rhodamine 110 label, whichis measured using 485 nm excitation and 530 nm detection.

In the experiment, 100 μl DHL-4 B-cells (1×10⁶ cells/ml) were plated inblack 96-well flat-bottom tissue culture plates and treated for 24 or 48hours with 1 μg/ml CD20×CD20 scorpion, Rituximab, an IgG controlantibody, or 5 μM staurosporine at 37° C., 5% CO₂ in a standardtissue-culture incubator. (Staurosporine is a small-molecule,broad-spectrum protein kinase inhibitor that is known in the art as apotent inducer of classical apoptosis in a wide variety of cell types.)After 24 or 48 hours, 100 μl of the 100-fold diluted substrate was addedto each well, gently mixed for one minute on a plate shaker (300 rpm)and incubated at room temperature for two hours. Fluorescence wasmeasured using 485 nM excitation and 527 nM emission filter (FluoroskanAscent FL, Thermo Labsystems). Graphs showing average fluorescentintensity of triplicate treatments plus/minus standard deviation after24 hours and 48 hours (24 hours only for staurosporine) are presented inFIG. 57. These results establish that CD20-binding scorpions do notdirectly kill B-cells by an apoptotic pathway involving activation ofcaspase 3/7.

The results obtained in the Apo-1 assay were confirmed by Western blotanalyses designed to detect pro-caspase cleavage resulting in activatedcaspase or to detect cleavage of PARP (Poly(ADP-Ribose) Polymerase), aprotein known to be cleaved by activated caspase 3. DHL-4 B-cells wereexposed to a CD20 binding scorpion or a control for 4, 24, or 48 hoursand cell lysates were fractionated on SDS-PAGE and blotted for Westernanalyses using conventional techniques. FIG. 58 presents the results inthe form of a collection of Western blots. The bottom three Westernsutilized anti-caspase antibodies to detect shifts in molecular weight ofthe caspase enzyme, reflecting proteolytic activation. For caspases 3,7, and 9, there was no evidence of caspase activation by any of theCD20-binding molecules. Staurosporine served as a positive control forthe assay, and induced pro-caspase cleavage to active caspase for eachof caspases 3, 7 and 9. The fourth Western blot shown in FIG. 58 revealsthat PARP, a known substrate of activated caspase 3, was not cleaved,consistent with a failure of CD20-binding scorpions to activate caspase3. The results of all of these experiments are consistent in showingthat caspase 3 activation is not a significant feature of the directcell killing of DHL-4 B-cells induced by CD20 binding scorpions.

In addition, a time series study was conducted to determine the effectof CD20 binding proteins, including a CD20×CD20 scorpion, on Caspase 3.DoHH2 or Su-DHL-6 B-cells were incubated with 10 nM CD20 binding protein(S0129 scorpion, 2Lm20-4 SMIP, or Rituxan®)+/−soluble CD16 Ig (40 nM),soluble CD16 Ig alone, or media. The cells were cultured in completeRPMI with 10% FBS at 3×10⁵/well/300 μl and harvested at 4 hours, 24hours or 72 hours. The 72-hour time-point samples were plated in 500 μlof the test agent. Cells were washed with PBS and then stained forintracellular active caspase-3 using the BD Pharmingen Caspase 3, ActiveForm, mAB Apoptosis Kit:FITC (cat no. 55048, BD Pharmingen, San Jose,Calif.). Briefly, after 2 additional washes in cold PBS, the cells weresuspended in cold cytofix/cytoperm solution and incubated on ice for 20minutes. Cells were then washed by centrifugation, aspirated, and washedtwo times with Perm/Wash buffer at room temperature. The samples werethen stained with 20 μl FITC-anti-caspase 3 in 100 μl of Perm-Washbuffer at room temperature in the dark for thirty minutes. The sampleswere then washed two times with Perm-Wash buffer, and resuspended in 500μl of Perm-Wash buffer. Washed cells were then transferred to FACs tubesand run on a FACs Calibur (BD Biosciences, San Jose, Calif.) andanalyzed with Cell Quest software (BD Biosciences, San Jose, Calif.).The results are shown in Table 16.

TABLE 16 Percentage Caspase-3 positive cells Percentage in live gateMolecule 48 48 (10 nM) 4 hours 24 hours hours 4 hours 24 hours hoursRTXN 7 25 7 75 53 56 and CD16 hi 27 47 21 79 60 43 (4X) CD20 SMIP 5 5 1089 85 81 (2Lm20-4) and CD16 hi 28 54 21 61 60 41 Humanized 7 13 14 69 6861 CD20xCD20 scorpion (S00129) and CD16 hi 30 31 15 67 75 72 Media 7 5 989 82 80 and CD16 hi 6 5 9 91 83 80

The results of all of these experiments are consistent in showing thatthere is limited activation of caspase 3 in the absence of CD16, whichdoes not implicate caspase 3 activation as a significant feature of thedirect cell killing induced by CD20 binding scorpions.

d. DNA Fragmentation

Induction of classical apoptotic signaling pathways ultimately resultsin condensation and fragmented degradation of chromosomal DNA. Todetermine whether CD20-binding scorpions directly killed B-cells througha classical apoptotic mechanism, the state of B-cell chromosomal DNA wasexamined following exposure of the cells to CD20-binding scorpions, orcontrols. Initially, DHL-4 B-cells were treated in vitro for 4, 24 or 48hours with a CD20-binding molecule, i.e., the monospecific CD20×CD20(2Lm20-4×2Lm20-4) scorpion, the CD20×CD20 (011×2Lm20-4) scorpion, orRituximab, or with a control. Subsequently, cells were lysed andchromosomal DNA was purified using conventional techniques. Thechromosomal DNA was then size-fractionated by gel electrophoresis. Thegel electrophoretogram shown in FIG. 59 reveals a lack of DNAfragmentation that demonstrated that the cell death generated byCD20-binding scorpions was not mediated by a classical apoptoticpathway. Staurosporine-treated cells were used as positive control inthese assays.

e. SYK Phosphorylation

SYK is a phospho-regulated protein with several phosphorylation sitesthat functions as a transcriptional repressor. SYK is localized to thecell nucleus, but is capable of rapid relocation to the membrane uponactivation. For activation, SYK must retain its nuclear localizationsequence. Activated SYK has a role in suppressing breast cancer tumorsand SYK is activated by pro-apoptotic signals such as ionizingradiation, BCR ligation and MHC class II cross-linking. Further, SYK hasbeen shown to affect the PLC-γ and Ca⁺⁺ pathways. Given theseobservations, the capacity of CD20-binding scorpions to affect SYK wasinvestigated.

DHL-6 B-cells were exposed to a bispecific CD20×CD37 scorpion for 0, 5,7 or 15 hours and the cells were lysed. Lysates were immunoprecipitatedwith either an anti-phosphotyrosine antibody or with an anti-SYKantibody. Immunoprecipitates were fractionated by gel electrophoresisand the results are shown in FIG. 60. Apparent from an inspection ofFIG. 60 is the failure of the bispecific CD20×CD37 scorpion to inducephosphorylation of SYK, thereby activating it. Consistent with theabove-described studies on caspase activation and chromosomal DNAfragmentation, it does not appear that CD20-binding scorpions directlykill B-cells using a classic apoptotic pathway, such as thecaspase-dependent pathway. While not wishing to be bound by theory, itis expected that the CD20-binding scorpions directly kill B-cellsthrough a caspase-, and SYK-, independent pathway that does notprominently feature chromosomal DNA fragmentation, at least not on thesame time frame as fragmentation occurs during caspase-dependentapoptosis.

Example 19 Scorpion Applications

a. In Vivo Activity of Scorpions

The activity of scorpions was also assessed using a mouse model.Measurements of scorpion activity in vivo involved administration of10-300 μg scorpion and subsequent time-series determinations of serumconcentrations of that scorpion. Results of these studies, presented asserum concentration curves for each of two bispecific scorpions (i.e.,S0033, a CD20×CD27 scorpion and a CD20×CD37 scorpion) from three-weekpharmacokinetic studies in mice are presented in FIG. 40. The data inFIG. 40 show that it took at least 500 hours after administration beforethe serum levels of each of the two bispecific scorpions fell back tobaseline levels. Thus, scorpions show serum stability and reproducible,sustained circulating half-lives in vivo.

The in vivo efficacy of scorpions was also assessed. An aggressive Ramosxenograft model was used in parallel experiments with SMIPs versushistorical immunoglobulin controls. The survival curves provided in FIG.41 reveal that administration of 10 μg bispecific scorpion hadnegligible influence on survival, but administration of 100-300 μg hadsignificant positive effect on the survival of mice bearing Ramosxenografts.

b. Combination Therapies

it is contemplated that scorpions will find application in theprevention, treatment or amelioration of a symptom of, a wide variety ofconditions affecting man, other mammals and other organisms. Forexample, CD20-binding scorpions are expected to be useful in treating orpreventing a variety of diseases associated with excessive or aberrantB-cells. In fact, any disease amenable to a treatment involving thedepletion of B-cells would be amenable to treatment with a CD20-bindingscorpion. In addition, scorpions, e.g., CD20-binding scorpions, may beused in combination therapies with other therapeutics. To illustrate thefeasibility of a wide variety of combination therapies, the monospecificCD20×CD20 scorpion (S0129) was administered to Su-DHL-6 B-cells incombination with doxorubicin, vincristine or rapamycin. Doxorubicin is atopoisomerase II poison that interferes with DNA biochemistry andbelongs to a class of drugs contemplated for anti-cancer treatment.Rapamycin (Sirolimus) is a macrolide antibiotic that inhibits theinitiation of protein synthesis and suppresses the immune system,finding application in organ transplantation and as ananti-proliferative used with coronary stents to inhibit or preventrestenosis. Vincristine is a vinca alkaloid that inhibits tubuleformation and has been used to treat cancer.

The experimental results shown in FIG. 61 are presented as CombinationIndex values for each combination over a range of effect levels. Theinteractions of the monospecific CD20×CD20 scorpion S0129 are differentfor each drug class, while with Rituxan® (RTXN) the plots forms aresimilar. The effect seen with doxorubicin at high concentrations mayreflect a shift towards monovalent binding. The data establish thatCD20-binding scorpions may be used in combination with a variety ofother therapeutics and such combinations would be apparent to one ofskill in the art in view of the present disclosure.

Variations on the structural themes for multivalent binding moleculeswith effector function, or scorpions, will be apparent to those of skillin the art upon review of the present disclosure, and such variantstructures are within the scope of the invention.

1-25. (canceled)
 26. A polypeptide comprising, from amino-terminus tocarboxy-terminus: (a) a first binding domain; (b) a constant sub-regioncomprising an immunoglobulin C_(H2) domain and a C_(H3) domain; (c) alinker peptide comprising an amino acid sequence from a stalk region ofa type II C-lectin protein; and (e) a second binding domain.
 27. Thepolypeptide of claim 26, wherein the first and/or second binding domaincomprises variable regions from an immunoglobulin.
 28. The polypeptideof claim 26, wherein the first and second binding domains specificallybind different target molecules located on the same cell.
 29. Thepolypeptide of claim 26, wherein the first and second binding domainsspecifically bind different target molecules located on physicallydistinct cells.
 30. The polypeptide of claim 26, wherein at least onebinding domain specifically binds a cell-free molecular target.
 31. Thepolypeptide of claim 26, wherein at least one binding domain is an scFvcomprising a sequence selected from SEQ ID NO:2, 4, 6, 103, 105, 107,and
 109. 32. The polypeptide of claim 26, wherein the type II C-lectinprotein is selected from the group consisting of CD69, CD72, CD94,NKG2A, and NKG2D.
 33. The polypeptide of claim 26, wherein the linkercomprises a sequence selected from SEQ ID NOS: 373, 374, 375, 376, and377.
 34. The polypeptide of claim 26, wherein the linker is at least 5amino acids in length.
 35. The polypeptide of claim 34, wherein thelinker is between 5 and 45 amino acids in length.
 36. The polypeptide ofclaim 26, wherein the linker is resistant to proteolytic cleavage. 37.The polypeptide of claim 27, wherein the first and/or second bindingdomains comprise chimeric, humanized, or human immunoglobulin variabledomains.
 38. The polypeptide of claim 26, wherein the constantsub-region comprises IgG1 immunoglobulin C_(H2) and C_(H3) domains. 39.The polypeptide of claim 26, wherein the constant sub-region comprisesC_(H2) and C_(H3) domains from a human immunoglobulin.
 40. Thepolypeptide of claim 26, wherein the polypeptide does not comprise aC_(H1) domain.
 41. The polypeptide of claim 26, wherein the constantsub-region further comprises an immunoglobulin hinge region.
 42. Thepolypeptide of claim 41, wherein the hinge region is a hinge regionselected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgE, IgA2,synthetic hinge and the hinge-like C_(H2) domain of IgM.
 43. Thepolypeptide of claim 42, wherein the hinge region is an IgG1 hingeregion.
 44. The polypeptide of claim 43, wherein the hinge region is ahuman IgG1 hinge region with a mutation at one or two cysteine residues.45. The polypeptide of claim 26, wherein at least one binding domainspecifically binds a target selected from a tumor antigen, a B-celltarget, a TNF receptor superfamily member, a Hedgehog family member, areceptor tyrosine kinase, a proteoglycan-related molecule, a TGF-betasuperfamily member, a Wnt-related molecule, a receptor ligand, a T-celltarget, a Dendritic cell target, an NK cell target, amonocyte/macrophage cell target, a myeloid target, and an angiogenesistarget.
 46. The polypeptide of claim 26, wherein one of the two bindingdomains specifically binds a target selected from CD3, CD20, CD37,CD79b, CD80, CD86, IRTA1, IRTA2, IRTA3, IRTA4, IRTA5, TNFRI/TNFRSF1A,TNFRII/TNFRSF1B, Fas/TNFRSF6, TRAILR/TNFRSF10, RANK/TNFRSF11A,Osteoprotegerin/TNFRSF11B, TWEAKR/TNFRSF12, HVEM/TNFRSF14,GITR/TNFRSF18, TNF-α/TNFSF1A, TNF-β/TNFSF1B, TRAIL/TNFSF10, FasLigand/TNFSF6, TWEAK/TNFSF12, APRIL/TNFSF13, LIGHT/TNFSF14,GITRL/TNFSF18, FGFR, Flt-3, HGFR, IGF-IR, IGF-IIR, MSPR/Ron, PDGFRα,PDGFRβ, EGFR, ErbB2, ErbB3, VEGFR1/Flt-1, VEGFR2/Flk-1, VEGFR3/Flt-4,TGF-βRI/ALK-5, TGF-βRII, TGF-βRIIb, EGF, TGF-α, IGF-I, IGF-II, BMP,TGF-β, FGF, P1GF, PDGF-A, PDGF-B, PDGF-C, PDGF-D, VEGF, VEGF-B, VEGF-C,VEGF-D, IL-2R, IL-2Rβ, IL-4R, B7-H3, IL-6R, IL-10Rα, IL-10Rβ, IL-12Rβ1,IL-12Rβ2, IL-13Rα1, Osteopontin, PD-1, CTLA-4, IFN-γ R1, IFN-γ R2,Receptor for Advanced Glycation End products (RAGE), IL-13, IL-22R,IL-21, and IL-4.
 47. The polypeptide of claim 26, wherein thepolypeptide comprises a binding domain pair specifically recognizing apair of targets selected from CD19/CD20, CD19/CD22, CD19/CL II,CD20/CD21, CD20/CD22, CD20/CD40, CD20/CD79a, CD20/CD79b, CD20/CD81,CD20/CL II, CD21/CD22, CD21/CD79b, CD21/CL II, CD22/CD23, CD22/CD30,CD22/CD37, CD22/CD40, CD22/CD70, CD22/CD72, CD22/CD79a, CD22/CD79b,CD22/CD80, CD22/CD86, CD22/CL II, CD23/CL II, CD30/CL II, CD37/CD79b,CD37/CL II, CD40/CD79b, CD40/CL II, CD70/CD79b, CD70/CL II, CD72/CD79b,CD72/CL II, CD79a/CD79b, CD79b/CD80, CD79b/CD81, CD79b/CD86, CD79b/CLII, CD80/CL II, and CD86/CL II.
 48. The polypeptide of claim 26, whereinthe immunoglobulin constant sub-region provides an effector functionselected from the group consisting of antibody-dependent cell-mediatedcytotoxicity, complement-dependent cytotoxicity, complement fixation,antibody-dependent cellular phagocytosis, binding to Fc receptors, andprotein A binding.
 49. The polypeptide of claim 26, wherein thepolypeptide has an extended in vivo half-life as compared to apolypeptide lacking the constant sub-region.
 50. The polypeptide ofclaim 26, wherein the polypeptide has an in vivo half-life of at least28 hours in a human.
 51. The polypeptide of claim 26, wherein thepolypeptide is capable of forming dimers.
 52. A nucleic acid encodingthe polypeptide of claim
 26. 53. A vector comprising the nucleic acid ofclaim
 52. 54. A host cell comprising the nucleic acid of claim
 52. 55. Ahost cell comprising the vector of claim
 53. 56. A compositioncomprising the polypeptide of claim 26 and a pharmaceutically acceptablecarrier.